James F. Conway

Polyglutamine disruption of the huntingtin exon 1 N terminus triggers a complex aggregation mechanism

Simple polyglutamine (polyQ) peptides aggregate in vitro via a nucleated growth pathway directly yielding amyloid-like aggregates. We show here that the 17-amino-acid flanking sequence (HTTNT) N-terminal to the polyQ in the toxic huntingtin exon 1 fragment imparts onto this peptide a complex alternative aggregation mechanism. In isolation, the HTTNT peptide is a compact coil that resists aggregation. When polyQ is fused to this sequence, it induces in HTTNT, in a repeat-length dependent fashion, a more extended conformation that greatly enhances its aggregation into globular oligomers with HTTNT cores and exposed polyQ. In a second step, a new, amyloid-like aggregate is formed with a core composed of both HTTNT and polyQ. The results indicate unprecedented complexity in how primary sequence controls aggregation within a substantially disordered peptide and have implications for the molecular mechanism of Huntingtons disease.

Molecular Tectonic Model of Virus Structural Transitions: the Putative Cell Entry States of Poliovirus

ABSTRACT Upon interacting with its receptor, poliovirus undergoes conformational changes that are implicated in cell entry, including the externalization of the viral protein VP4 and the N terminus of VP1. We have determined the structures of native virions and of two putative cell entry intermediates, the 135S and 80S particles, at ∼22-Å resolution by cryo-electron microscopy. The 135S and 80S particles are both ∼4% larger than the virion. Pseudoatomic models were constructed by adjusting the beta-barrel domains of the three capsid proteins VP1, VP2, and VP3 from their known positions in the virion to fit the 135S and 80S reconstructions. Domain movements of up to 9 Å were detected, analogous to the shifting of tectonic plates. These movements create gaps between adjacent subunits. The gaps at the sites where VP1, VP2, and VP3 subunits meet are plausible candidates for the emergence of VP4 and the N terminus of VP1. The implications of these observations are discussed for models in which the externalized components form a transmembrane pore through which viral RNA enters the infected cell.

Intermediate filament structure: 3. Analysis of sequence homologies

Abstract Homology profiles have been calculated for each chain classification (Types I–V) using all available IF sequence data and detailed analyses of the results have been made. Features of structural relevance which have been noted include the following: (i) The linear distribution of homology scores for the acidic and the basic residues in segments 1B and 2 includes the same periods as those previously observed in the linear distribution of the same residue types in individual chains. This confirms that the spatial distribution of the ionic residues has been maintained during evolution, probably as a consequence of its importance in specifying the aggregation of molecules in IF. (ii) The maintenance of highly conserved sequences at the ends of the rod domain appears crucial to IF structure in general but significant differences in the location of regions of very high (or very low) homology are apparent when one chain type is compared to another. Furthermore, although differences exist between the various types of IF chains, the pronounced homologous features argue that intermediate filaments are a polymorphic family of closely related structures. (iii) The high degree of homology in the L2 link segment in all chain types suggests a highly conserved structure, probably α-helical; a limited degree of homology in all chain types for segment L12 is consistent with a partially conserved structure, possibly β; the highly variable length of link L1, together with the low level of homology present, suggests an irregular conformation. (iv) On the basis of sequence homology the Type I and Type II sequences can each be subdivided. Type Ia and IIa chains constitute those from the ‘hard’ α-keratins and Type Ib and IIb chains constitute those from the ‘soft’ epidermal keratins. (v) The neurofilament chains are confirmed as Type IV chains and not Type III. (vi) The NF-L, NF-M and NF-H neurofilament chains may be clearly distinguished within the Type IV classification on the basis of homology within both the rod domain and the H1 and H2 subdomains. (vii) The extended sequence data base now available has revealed that both the H1 and H2 subdomains differ significantly in extent. For example, the H1 subdomain encompasses the entire N-terminal domain of Type IV chains (both NF-L and NF-M) and the H2 subdomain encompasses the entire C-terminal domain of Type III chains. In contrast the Type I IF chains appear to have negligible H subdomains.

Structural features in the heptad substructure and longer range repeats of two-stranded α-fibrous proteins

Considerable sequence data have been collected from the intermediate filament proteins and other alpha-fibrous proteins including myosin, tropomyosin, paramyosin, desmoplakin and M-protein. The data show that there is a clear preference for some amino acids to occur in specific positions within the heptad substructure that characterizes the sequences which form the coiled-coil rod domain in this class of proteins. The results also indicate that although there are major similarities between the various proteins there are also key differences. In all cases, however, significant regularities in the linear disposition of the acidic and the basic residues in the coiled-coil segments can be related to modes of chain and molecular aggregation. In particular a clear trend has been observed which relates the mode of molecular aggregation to the number of interchain ionic interactions per heptad pair.

Novel fold and capsid-binding properties of the |[lambda]|-phage display platform protein gpD

The crystal structure of gpD, the capsid-stabilizing protein of bacteriophage λ, was solved at 1.1 Å resolution. Data were obtained from twinned crystals in space group P21 and refined with anisotropic temperature factors to an R-factor of 0.098 (Rfree = 0.132). GpD (109 residues) has a novel fold with an unusually low content of regular secondary structure. Noncrystallographic trimers with substantial intersubunit interfaces were observed. The C-termini are well ordered and located on one side of the trimer, relatively far from its three-fold axis. The N-termini are disordered up to Ser 15, which is close to the three-fold axis and on the same side as the C-termini. A density map of the icosahedral viral capsid at 15 Å resolution, obtained by cryo-electron microscopy and image reconstruction, reveals gpD trimers, seemingly indistinguishable from the ones seen in the crystals, at all three-fold sites. The map further reveals that the side of the trimer that binds to the capsid is the side on which both termini reside. Despite this orientation of the gpD trimer, fusion proteins connected by linker peptides to either terminus bind to the capsid, allowing protein and peptide display.

A Quasi-Atomic Model of Human Adenovirus Type 5 Capsid.

Adenoviruses infect a wide range of vertebrates including humans. Their icosahedral capsids are composed of three major proteins: the trimeric hexon forms the facets and the penton, a noncovalent complex of the pentameric penton base and trimeric fibre proteins, is located at the 12 capsid vertices. Several proteins (IIIa, VI, VIII and IX) stabilise the capsid. We have obtained a 10 Å resolution map of the human adenovirus 5 by image analysis from cryo‐electron micrographs (cryoEMs). This map, in combination with the X‐ray structures of the penton base and hexon, was used to build a quasi‐atomic model of the arrangement of the two major capsid components and to analyse the hexon–hexon and hexon–penton interactions. The secondary proteins, notably VIII, were located by comparing cryoEM maps of native and pIX deletion mutant virions. Minor proteins IX and IIIa are located on the outside of the capsid, whereas protein VIII is organised with a T=2 lattice on the inner face of the capsid. The capsid organisation is compared with the known X‐ray structure of bacteriophage PRD1.

The cellular receptor to human rhinovirus 2 binds around the 5-fold axis and not in the canyon: a structural view

Human rhinovirus serotype 2 (HRV2) belongs to the minor group of HRVs that bind to members of the LDL‐receptor family including the very low density lipoprotein (VLDL)‐receptor (VLDL‐R). We have determined the structures of the complex between HRV2 and soluble fragments of the VLDL‐R to 15 Å resolution by cryo‐electron microscopy. The receptor fragments, which include the first three ligand‐binding repeats of the VLDL‐R (V1–3), bind to the small star‐shaped dome on the icosahedral 5‐fold axis. This is in sharp contrast to the major group of HRVs where the receptor site for ICAM‐1 is located at the base of a depression around each 5‐fold axis. Homology models of the three domains of V1–3 were used to explore the virus–receptor interaction. The footprint of VLDL‐R on the viral surface covers the BC‐ and HI‐loops on VP1.

Hierarchical self-assembly of amelogenin and the regulation of biomineralization at the nanoscale

Enamel is a highly organized hierarchical nanocomposite, which consists of parallel arrays of elongated apatitic crystallites forming an intricate three-dimensional microstructure. Amelogenin, the major extracellular matrix protein of dental enamel, regulates the formation of these crystalline arrays via cooperative interactions with forming mineral phase. Using cryoelectron microscopy, we demonstrate that amelogenin undergoes stepwise hierarchical self-assembly. Furthermore, our results indicate that interactions between amelogenin hydrophilic C-terminal telopeptides are essential for oligomer formation and for subsequent steps of hierarchical self-assembly. We further show that amelogenin assemblies stabilize mineral prenucleation clusters and guide their arrangement into linear chains that organize as parallel arrays. The prenucleation clusters subsequently fuse together to form needle-shaped mineral particles, leading to the formation of bundles of crystallites, the hallmark structural organization of the forming enamel at the nanoscale. These findings provide unique insight into the regulation of biological mineralization by specialized macromolecules and an inspiration for bottom-up strategies for the materials design.

An Estimate of the Mean Length of Collagen Fibrils in Rat Tail-Tendon as a Function of age

A theoretical expression has been derived for the mean collagen fibril length in tendon based on the assumption that collagen fibrils originate in cell surface invaginations and terminate either at some remote cell surface or another collagen fibril bundle. The expression thus determined requires knowledge of the effective lengths of the fibrocytes (or fibrocyte assemblies) and the cellular content of the tendon. Both of these parameters have been measured experimentally as a function of age for rat-tail tendon using a combined light microscope and electron microscope approach. The results obtained for immature tendon suggest that the mean collagen fibril length is at least equal to the critical length required to maintain the appropriate tensile properties. In the most mature tissue studied, however, the mean-collagen fibril length is in excess of 100 times the critical length.

The Herpes Simplex Virus 1 UL17 Protein Is the Second Constituent of the Capsid Vertex-Specific Component Required for DNA Packaging and Retention

ABSTRACT The herpes simplex virus (HSV) UL17 and UL25 minor capsid proteins are essential for DNA packaging. They are thought to comprise a molecule arrayed in five copies around each of the capsid vertices. This molecule was initially termed the “C-capsid-specific component” (CCSC) (B. L. Trus et al., Mol. Cell 26:479-489, 2007), but as we have subsequently observed this feature on reconstructions of A, B, and C capsids, we now refer to it more generally as the “capsid vertex-specific component” (CVSC) (S. K. Cockrell et al., J. Virol. 85:4875-4887, 2011). We previously confirmed that UL25 occupies the vertex-distal region of the CVSC density by visualizing a large UL25-specific tag in reconstructions calculated from cryo-electron microscopy (cryo-EM) images. We have pursued the same strategy to determine the capsid location of the UL17 protein. Recombinant viruses were generated that contained either a small tandem affinity purification (TAP) tag or the green fluorescent protein (GFP) attached to the C terminus of UL17. Purification of the TAP-tagged UL17 or a similarly TAP-tagged UL25 protein clearly demonstrated that the two proteins interact. A cryo-EM reconstruction of capsids containing the UL17-GFP protein reveals that UL17 is the second component of the CVSC and suggests that UL17 interfaces with the other CVSC component, UL25, through its C terminus. The portion of UL17 nearest the vertex appears to be poorly constrained, which may provide flexibility in interacting with tegument proteins or the DNA-packaging machinery at the portal vertex. The exposed locations of the UL17 and UL25 proteins on the HSV-1 capsid exterior suggest that they may be attractive targets for highly specific antivirals.