Stewart R. Durell

The HIV Env-mediated fusion reaction

The current general model of HIV viral entry involves the binding of the trimeric viral envelope glycoprotein gp120/gp41 to cell surface receptor CD4 and chemokine co-receptor CXCR4 or CCR5, which triggers conformational changes in the envelope proteins. Gp120 then dissociates from gp41, allowing for the fusion peptide to be inserted into the target membrane and the pre-hairpin configuration of the ectodomain to form. The C-terminal heptad repeat region and the leucine/isoleucine zipper region then form the thermostable six-helix coiled-coil, which drives the membrane merger and eventual fusion. This model needs updating, as there has been a wealth of data produced in the last few years concerning HIV entry, including target cell dependencies, fusion kinetic data, and conformational intermediates. A more complete model must include the involvement of membrane microdomains, actin polymerization, glycosphingolipids, and possibly CD4 and chemokine signaling in entry. In addition, kinetic experiments involving the addition of fusion inhibitors have revealed some of the rate-limiting steps in this process, adding a temporal component to the model. A review of these data that may require an updated version of the original model is presented here.

Epitope Mapping of CCR5 Reveals Multiple Conformational States and Distinct but Overlapping Structures Involved in Chemokine and Coreceptor Function

The chemokine receptor CCR5 is the major coreceptor for R5 human immunodeficiency virus type-1 strains. We mapped the epitope specificities of 18 CCR5 monoclonal antibodies (mAbs) to identify domains of CCR5 required for chemokine binding, gp120 binding, and for inducing conformational changes in Env that lead to membrane fusion. We identified mAbs that bound to N-terminal epitopes, extracellular loop 2 (ECL2) epitopes, and multidomain (MD) epitopes composed of more than one single extracellular domain. N-terminal mAbs recognized specific residues that span the first 13 amino acids of CCR5, while nearly all ECL2 mAbs recognized residues Tyr-184 to Phe-189. In addition, all MD epitopes involved ECL2, including at least residues Lys-171 and Glu-172. We found that ECL2-specific mAbs were more efficient than NH2- or MD-antibodies in blocking RANTES or MIP-1β binding. By contrast, N-terminal mAbs blocked gp120-CCR5 binding more effectively than ECL2 mAbs. Surprisingly, ECL2 mAbs were more potent inhibitors of viral infection than N-terminal mAbs. Thus, the ability to block virus infection did not correlate with the ability to block gp120 binding. Together, these results imply that chemokines and Env bind to distinct but overlapping sites in CCR5, and suggest that the N-terminal domain of CCR5 is more important for gp120 binding while the extracellular loops are more important for inducing conformational changes in Env that lead to membrane fusion and virus infection. Measurements of individual antibody affinities coupled with kinetic analysis of equilibrium binding states also suggested that there are multiple conformational states of CCR5. A previously described mAb, 2D7, was unique in its ability to effectively block both chemokine and Env binding as well as coreceptor activity. 2D7 bound to a unique antigenic determinant in the first half of ECL2 and recognized a far greater proportion of cell surface CCR5 molecules than the other mAbs examined. Thus, the epitope recognized by 2D7 may represent a particularly attractive target for CCR5 antagonists.

Evolutionary Relationship between K+ Channels and Symporters

The hypothesis is presented that at least four families of putative K(+) symporter proteins, Trk and KtrAB from prokaryotes, Trk1,2 from fungi, and HKT1 from wheat, evolved from bacterial K(+) channel proteins. Details of this hypothesis are organized around the recently determined crystal structure of a bacterial K(+) channel: i. e., KcsA from Streptomyces lividans. Each of the four identical subunits of this channel has two fully transmembrane helices (designated M1 and M2), plus an intervening hairpin segment that determines the ion selectivity (designated P). The symporter sequences appear to contain four sequential M1-P-M2 motifs (MPM), which are likely to have arisen from gene duplication and fusion of the single MPM motif of a bacterial K(+) channel subunit. The homology of MPM motifs is supported by a statistical comparison of the numerical profiles derived from multiple sequence alignments formed for each protein family. Furthermore, these quantitative results indicate that the KtrAB family of symporters has remained closest to the single-MPM ancestor protein. Strong sequence evidence is also found for homology between the cytoplasmic C-terminus of numerous bacterial K(+) channels and the cytoplasm-resident TrkA and KtrA subunits of the Trk and KtrAB symporters, which in turn are homologous to known dinucleotide-binding domains of other proteins. The case for homology between bacterial K(+) channels and the four families of K(+) symporters is further supported by the accompanying manuscript, in which the patterns of residue conservation are demonstrated to be similar to each other and consistent with the known 3D structure of the KcsA K(+) channel.

Theoretical models of the ion channel structure of amyloid beta-protein.

Theoretical methods are used to develop models for the ion channel structure of the membrane-bound amyloid beta-protein. This follows recent observations that the beta-protein forms cation-selective channels in lipid bilayers in vitro. Amyloid beta-protein is the main component of the extracellular plaques in the brain that are characteristic of Alzheimers disease. Based on the amino acid sequence and the unique environment of the membrane, the secondary structure of the 40-residue beta-protein is predicted to form a beta-hairpin followed by a helix-turn-helix motif. The channel structures were-designed as aggregates of peptide subunits in identical conformations. Three types of models were developed that are distinguished by whether the pore is formed by the beta-hairpins, the middle helices, or by the more hydrophobic C-terminal helices. The latter two types can be converted back and forth by a simple conformational change, which would explain the variable conduction states observed for a single channel. It is also demonstrated how lipid headgroups could be incorporated into the pore lining, and thus affect the ion selectivity. The atomic-scale detail of the models make them useful for designing experiments to determine the real structure of the channel, and thus further the understanding of peptide channels in general. In addition, if beta-protein-induced channel activity is found to be the cause of cell death in Alzheimers disease, then the models may be helpful in designing counteracting drugs.

Magainin 2, a natural antibiotic from frog skin, forms ion channels in lipid bilayer membranes

We have examined the ion channel forming properties of magainin 2 by incorporating the peptide into artificial lipid bilayers held under voltage clamp. Magainin 2 increased lipid bilayer conductance in a concentration dependent manner with a Hill coefficient of 1.7. The magainin 2 conductance was selective for monovalent cations over anions with a ratio of 5:1 and had both voltage-sensitive and -insensitive components. Two structurally related but antibiotically less potent analogues, magainin 1 and Z-12, also increased lipid bilayer conductance with a similar ion selectivity but these peptides were less potent than magainin 2. We propose that the weak cation selectivity of the magainin channels can be accounted for by the inclusion of negatively charged lipids in the channel complex and suggest two possible structures for such a channel. The ionophoric properties of these peptides are likely to be proximal to their antibiotic activities.

STRUCTURAL MODELS OF THE KTRB, TRKH, AND TRK1,2 SYMPORTERS BASED ON THE STRUCTURE OF THE KCSA K+ CHANNEL

Three-dimensional computer modeling is used to further investigate the hypothesis forwarded in the accompanying paper of an evolutionary relationship between four related families of K(+) sympoter proteins and the superfamily of K(+) channel proteins. Atomic-scale models are developed for the transmembrane regions of one member from each of the three more distinct symporter families, i.e., a TrkH protein from Escherichia coli, a KtrB protein from Aquifex aeolicus, and a Trk1,2 protein from Schizosaccharomyces pombe. The portions of the four consecutive M1-P-M2 motifs in the symporters that can be aligned with K(+) channel sequences are modeled directly from the recently determined crystal structure of the KcsA K(+) channel from Streptomyces lividans. The remaining portions are developed using our previously accumulated theoretical modeling criteria and principles. Concurrently, the use of these criteria and principles is further supported by the now verified predictions of our previous K(+) channel modeling efforts and the degree to which they are satisfied by the known structure of the KcsA protein. Thus the observed ability of the portions of the symporter models derived from the KcsA crystal structure to also satisfy the theoretical modeling criteria provides additional support for an evolutionary link with K(+) channel proteins. Efforts to further satisfy the criteria and principles suggest that the symporter proteins from fungi and plants (i.e., Trk1,2 and HKT1) form dimeric and/or tetrameric complexes in the membrane. Furthermore, analysis of the atomic-scale models in relation to the sequence conservation within and between the protein families suggests structural details for previously proposed mechanisms for the linked symport of K(+) with Na(+) and H(+). Suggestions are also given for experiments to test these structures and hypotheses.

Relating molecular flexibility to function: a case study of tubulin.

Microtubules (MT), along with a variety of associated motor proteins, are involved in a range of cellular functions including vesicle movement, chromosome segregation, and cell motility. MTs are assemblies of heterodimeric proteins, alpha beta-tubulins, the structure of which has been determined by electron crystallography of zinc-induced, pacilitaxel-stabilized tubulin sheets. These data provide a basis for examining relationships between structural features and protein function. Here, we study the fluctuation dynamics of the tubulin dimer with the aim of elucidating its functional motions relevant to substrate binding, polymerization/depolymerization and MT assembly. A coarse-grained model, harmonically constrained according to the crystal structure, is used to explore the global dynamics of the dimer. Our results identify six regions of collective motion, comprised of structurally close but discontinuous sequence fragments, observed only in the dimeric form, dimerization being a prerequisite for domain identification. Boundaries between regions of collective motions appear to act as linkages, found primarily within secondary-structure elements that lack sequence conservation, but are located at minima in the fluctuation curve, at positions of hydrophobic residues. Residue fluctuations within these domains identify the most mobile regions as loops involved in recognition of the adjacent regions. The least mobile regions are associated with nucleotide binding sites where lethal mutations occur. The functional coupling of motions between and within regions identifies three global motions: torsional and wobbling movements, en bloc, between the alpha- and beta-tubulin monomers, and stretching longitudinally. Further analysis finds the antitumor drug pacilitaxel (TaxotereR) to reduce flexibility in the M loop of the beta-tubulin monomer; an effect that may contribute to tightening lateral interactions between protofilaments assembled into MTs. Our analysis provides insights into relationships between intramolecular tubulin movements of MT organization and function.

HIV Entry and Envelope Glycoprotein-mediated Fusion

HIV entry involves binding of the trimeric viral envelope glycoprotein (Env) gp120/gp41 to cell surface receptors, which triggers conformational changes in Env that drive the membrane fusion reaction. The conformational landscape that the lipids and Env navigate en route to fusion has been examined by biophysical measurements on the microscale, whereas electron tomography, x-rays, and NMR have provided insights into the process on the nanoscale and atomic scale. However, the coupling between the lipid and protein pathways that give rise to fusion has not been resolved. Here, we discuss the known and unknown about the overall HIV Env-mediated fusion process.

Modeling the ion channel structure of cecropin

Atomic-scale computer models were developed for how cecropin peptides may assemble in membranes to form two types of ion channels. The models are based on experimental data and physiochemical principles. Initially, cecropin peptides, in a helix-bend-helix motif, were arranged as antiparallel dimers to position conserved residues of adjacent monomers in contact. The dimers were postulated to bind to the membrane with the NH2-terminal helices sunken into the head-group layer and the COOH-terminal helices spanning the hydrophobic core. This causes a thinning of the top lipid layer of the membrane. A collection of the membrane bound dimers were then used to form the type I channel structure, with the pore formed by the transmembrane COOH-terminal helices. Type I channels were then assembled into a hexagonal lattice to explain the large number of peptides that bind to the bacterium. A concerted conformational change of a type I channel leads to the larger type II channel, in which the pore is formed by the NH2-terminal helices. By having the dimers move together, the NH2-terminal helices are inserted into the hydrophobic core without having to desolvate the charged residues. It is also shown how this could bring lipid head-groups into the pore lining.

Structural Basis for p300 Taz2/p53 TAD1 Binding and Modulation by Phosphorylation

Coactivators CREB-binding protein and p300 play important roles in mediating the transcriptional activity of p53. Until now, however, no detailed structural information has been available on how any of the domains of p300 interact with p53. Here, we report the NMR structure of the complex of the Taz2 (C/H3) domain of p300 and the N-terminal transactivation domain of p53. In the complex, p53 forms a short alpha helix and interacts with the Taz2 domain through an extended surface. Mutational analyses demonstrate the importance of hydrophobic residues for complex stabilization. Additionally, they suggest that the increased affinity of Taz2 for p53(1-39) phosphorylated at Thr(18) is due in part to electrostatic interactions of the phosphate with neighboring arginine residues in Taz2. Thermodynamic experiments revealed the importance of hydrophobic interactions in the complex of Taz2 with p53 phosphorylated at Ser(15) and Thr(18).