Richard B. Sessions

Self-assembling cages from coiled-coil peptide modules

From Coils to Cages Self-assembly strategies that mimic protein assembly, such as the formation of viral coats, often begin with simpler peptide assemblies. Fletcher et al. (p. 595, published online 11 April; see the Perspective by Ardejani and Orner) designed two coiled-coil peptide motifs, a heterodimer, and a homotrimer. Both peptides contained cysteine residues and could link through disulfide bonds, so that the trimer could form the vertices of a hexagonal network and the dimer its edges. However, these components are flexible and, rather than form extended sheets, they closed to form particles ∼100 nanometers in diameter. Hexagonal networks form from heterodimeric and homotrimeric coiled coils and create ~100-nanometer-diameter cages. [Also see Perspective by Ardejani and Orner] An ability to mimic the boundaries of biological compartments would improve our understanding of self-assembly and provide routes to new materials for the delivery of drugs and biologicals and the development of protocells. We show that short designed peptides can be combined to form unilamellar spheres approximately 100 nanometers in diameter. The design comprises two, noncovalent, heterodimeric and homotrimeric coiled-coil bundles. These are joined back to back to render two complementary hubs, which when mixed form hexagonal networks that close to form cages. This design strategy offers control over chemistry, self-assembly, reversibility, and size of such particles.

Interaction between prion protein and toxic amyloid β assemblies can be therapeutically targeted at multiple sites

A role for PrP in the toxic effect of oligomeric forms of Aβ, implicated in Alzheimers disease (AD), has been suggested but remains controversial. Here we show that PrP is required for the plasticity-impairing effects of ex vivo material from human AD brain and that standardized Aβ-derived diffusible ligand (ADDL) preparations disrupt hippocampal synaptic plasticity in a PrP-dependent manner. We screened a panel of anti-PrP antibodies for their ability to disrupt the ADDL–PrP interaction. Antibodies directed to the principal PrP/Aβ-binding site and to PrP helix-1, were able to block Aβ binding to PrP suggesting that the toxic Aβ species are of relatively high molecular mass and/or may bind multiple PrP molecules. Two representative and extensively characterized monoclonal antibodies directed to these regions, ICSM-35 and ICSM-18, were shown to block the Aβ-mediated disruption of synaptic plasticity validating these antibodies as candidate therapeutics for AD either individually or in combination.

Computational design of water-soluble α-helical barrels

Building with alphahelical coiled coils Understanding how proteins fold into well-defined three-dimensional structures has been a longstanding challenge. Increased understanding has led to increased success at designing proteins that mimic existing protein folds. This raises the possibility of custom design of proteins with structures not seen in nature. Thomson et al. describe the design of channelcontaining α-helical barrels, and Huang et al. designed hyperstable helical bundles. Both groups used rational and computational design to make new protein structures based on α-helical coiled coils but took different routes to reach different target structures. Science, this issue p. 485, p. 481 Protein design expands the repertoire of coiled-coil structures to α-helical barrels and hyperstable helical bundles. The design of protein sequences that fold into prescribed de novo structures is challenging. General solutions to this problem require geometric descriptions of protein folds and methods to fit sequences to these. The α-helical coiled coils present a promising class of protein for this and offer considerable scope for exploring hitherto unseen structures. For α-helical barrels, which have more than four helices and accessible central channels, many of the possible structures remain unobserved. Here, we combine geometrical considerations, knowledge-based scoring, and atomistic modeling to facilitate the design of new channel-containing α-helical barrels. X-ray crystal structures of the resulting designs match predicted in silico models. Furthermore, the observed channels are chemically defined and have diameters related to oligomer state, which present routes to design protein function.

A de novo peptide hexamer with a mutable channel

The design of new proteins that expand the repertoire of natural protein structures represents a formidable challenge. Success in this area would increase understanding of protein structure, and present new scaffolds that could be exploited in biotechnology and synthetic biology. Here we describe the design, characterisation and X-ray crystal structure of a new coiled-coil protein. The de novo sequence forms a stand-alone, parallel, 6-helix bundle with a channel running through it. Although lined exclusively by hydrophobic leucine and isoleucine side chains, the 6 Å channel is permeable to water. One layer of leucine residues within the channel is mutable accepting polar aspartic acid (Asp) and histidine (His) side chains, and leading to subdivision and organization of solvent within the lumen. Moreover, these mutants can be combined to form a stable and unique (Asp-His)3 heterohexamer. These new structures provide a basis for engineering de novo proteins with new functions.

Tyrphostin A23 inhibits internalization of the transferrin receptor by perturbing the interaction between tyrosine motifs and the medium chain subunit of the AP-2 adaptor complex

Several intracellular membrane trafficking events are mediated by tyrosine-containing motifs within the cytosolic domains of integral membrane proteins. Many such motifs conform to the consensus YXXΦ, where Φ represents a bulky hydrophobic residue. This motif interacts with the medium chain (μ) subunits of adaptor complexes that link the cytosolic domains of integral membrane proteins to the clathrin coat involved in vesicle formation. The YXXΦ motif is similar to motifs in which the tyrosine residue is phosphorylated by tyrosine kinases. Tyrphostins (structural analogs of tyrosine) are inhibitors of tyrosine kinases and function by binding to the active sites of the enzymes. We previously showed that, in vitro and in yeast two-hybrid interaction assays, some tyrphostins can inhibit the interaction between YXXΦ motifs and the μ2 subunit of the AP-2 adaptor complex (Crump, C., Williams, J. L., Stephens, D. J., and Banting, G. (1998) J. Biol. Chem. 273, 28073–28077). A23 is such a tyrphostin. We now show that molecular modeling of tyrphostin A23 into the tyrosine-binding pocket in μ2 provides a structural explanation for A23 being able to inhibit the interaction between YXXΦ motifs and μ2. Furthermore, we show that A23 inhibited the internalization of125I-transferrin in Heb7a cells without having any discernible effect on the morphology of compartments of the endocytic pathway. Control tyrphostins, active as inhibitors of tyrosine kinase activity, but incapable of inhibiting the YXXΦ motif/μ2 interaction, did not inhibit endocytosis. These data are consistent with A23 inhibition of the YXXΦ motif/μ2 interaction in intact cells and with the possibility that different tyrphostins may be used to inhibit specific membrane trafficking events in eukaryotic cells.

Molecular basis for SNX-BAR-mediated assembly of distinct endosomal sorting tubules.

Sorting nexins (SNXs) are regulators of endosomal sorting. For the SNX‐BAR subgroup, a Bin/Amphiphysin/Rvs (BAR) domain is vital for formation/stabilization of tubular subdomains that mediate cargo recycling. Here, by analysing the in vitro membrane remodelling properties of all 12 human SNX‐BARs, we report that some, but not all, can elicit the formation of tubules with diameters that resemble sorting tubules observed in cells. We reveal that SNX‐BARs display a restricted pattern of BAR domain‐mediated dimerization, and by resolving a 2.8 Å structure of a SNX1‐BAR domain homodimer, establish that dimerization is achieved in part through neutralization of charged residues in the hydrophobic BAR‐dimerization interface. Membrane remodelling also requires functional amphipathic helices, predicted to be present in all SNX‐BARs, and the formation of high order SNX‐BAR oligomers through selective ‘tip–loop’ interactions. Overall, the restricted and selective nature of these interactions provide a molecular explanation for how distinct SNX‐BAR‐decorated tubules are nucleated from the same endosomal vacuole, as observed in living cells. Our data provide insight into the molecular mechanism that generates and organizes the tubular endosomal network.

Crystal Structure of Streptococcus pyogenes Sortase A: Implications for Sortase mechanism

Sortases are a family of Gram-positive bacterial transpeptidases that anchor secreted proteins to bacterial cell surfaces. These include many proteins that play critical roles in the virulence of Gram-positive bacterial pathogens such that sortases are attractive targets for development of novel antimicrobial agents. All Gram-positive pathogens express a “housekeeping” sortase that recognizes the majority of secreted proteins containing an LPXTG wall-sorting motif and covalently attaches these to bacterial cell wall peptidoglycan. Many Gram-positive pathogens also express additional sortases that link a small number of proteins, often with variant wall-sorting motifs, to either other surface proteins or peptidoglycan. To better understand the mechanisms of catalysis and substrate recognition by the housekeeping sortase produced by the important human pathogen Streptococcus pyogenes, the crystal structure of this protein has been solved and its transpeptidase activity established in vitro. The structure reveals a novel arrangement of key catalytic residues in the active site of a sortase, the first that is consistent with kinetic analysis. The structure also provides a complete description of residue positions surrounding the active site, overcoming the limitation of localized disorder in previous structures of sortase A-type proteins. Modification of the active site Cys through oxidation to its sulfenic acid form or by an alkylating reagent supports a role for a reactive thiol/thiolate in the catalytic mechanism. These new insights into sortase structure and function could have important consequences for inhibitor design.

A designed system for assessing how sequence affects α to β conformational transitions in proteins

The role of amino acid sequence in conformational switching observed in prions and proteins associated with amyloid diseases is not well understood. To study α to β conformational transitions, we designed a series of peptides with structural duality; namely, peptides with sequence features of both an α−helical leucine zipper and a β−hairpin. The parent peptide, Template−α, was designed to be a canonical leucine-zipper motif and was confirmed as such using circular dichroism spectroscopy and analytical ultracentrifugation. To introduce β−structure character into the peptide, glutamine residues at sites away from the leucine-zipper dimer interface were replaced by threonine to give Template−αT. Unlike the parent peptide, Template−αT underwent a heat-inducible switch to β−structure, which reversibly formed gels containing amyloid-like fibrils. In contrast to certain other natural proteins where destabilization of the native states facilitate transitions to amyloid, destabilization of the leucine-zipper form of Template−αT did not promote a transformation. Cross-linking the termini of the peptides compatible with the alternative β−hairpin design, however, did promote the change. Furthermore, despite screening various conditions, only the internally cross-linked form of the parent, Template−α, peptide formed amyloid-like fibrils. These findings demonstrate that, in addition to general properties of the polypeptide backbone, specific residue placements that favor β−structure promote amyloid formation.

Designed α-Helical Tectons for Constructing Multicomponent Synthetic Biological Systems

One possible route to develop new synthetic-biological systems is to assemble discrete nanoscale objects from programmed peptide-based building blocks. We describe an algorithm to design such blocks based on the coiled-coil protein-folding motif. The success of the algorithm is demonstrated by the production of six peptides that form three target parallel, blunted-ended heterodimers in preference to any of the other promiscuous pairings and alternate configurations, for example, homodimers, sticky-ended assemblies, and antiparallel arrangements. The peptides were linked to promote the assembly of larger, defined nanoscale rods, thus demonstrating that targeted peptide-peptide interactions can be specified in complex mixtures.

Protochlorophyllide oxidoreductase: a homology model examined by site-directed mutagenesis.

An homology model of protochlorophyllide reductase (POR) from Synechocystis sp. was constructed on a template from the tyrosine‐dependent oxidoreductase family. The model showed characteristics appropriate to a globular, soluble protein and was used to generate a structure of the ternary complex of POR, nicotinamide adenine dinucleotide phosphate (NADPH), and protochlorophyllide. The POR ternary model was validated by mutagenesis experiments involving predicted coenzyme‐binding residues and by chemical modification experiments. A core tryptophan residue was shown to be responsible for much of the proteins fluorescence. Both quenching of this residue by coenzyme and fluorescence resonance energy transfer (FRET) from the protein to the coenzyme allowed the binding constant of NADPH to be determined. Replacement of this residue by Tyr gave an active mutant with approximately halved fluorescence and a negligible FRET signal, consistent with the role of this residue in energy transfer to the NADPH at the active site and with the model. The mechanism of the enzyme is discussed in the context of the model and semiempirical molecular orbital calculations. Proteins 2001;44:329–335.