Structure of the protective nematode protease complex H-gal-GP and its conservation across roundworm parasites.

Roundworm parasite infections are a major cause of human and livestock disease worldwide and a threat to global food security. Disease control currently relies on anthelmintic drugs to which roundworms are becoming increasingly resistant. An alternative approach is control by vaccination and 'hidden antigens', components of the worm gut not encountered by the infected host, have been exploited to produce Barbervax, the first commercial vaccine for a gut dwelling nematode of any host. Here we present the structure of H-gal-GP, a hidden antigen from Haemonchus contortus, the Barber's Pole worm, and a major component of Barbervax. We demonstrate its novel architecture, subunit composition and topology, flexibility and heterogeneity using cryo-electron microscopy, mass spectrometry, and modelling. Importantly, we demonstrate that complexes with the same architecture are present in other Strongylid roundworm parasites including human hookworm. This suggests a common ancestry and the potential for development of a unified hidden antigen vaccine.

The mitosis and neurodevelopment proteins NDE1 and NDEL1 form dimers, tetramers, and polymers with a folded back structure in solution.

Paralogs NDE1 (nuclear distribution element 1) and NDEL1 (NDE-like 1) are essential for mitosis and neurodevelopment. Both proteins are predicted to have similar structures, based upon high sequence similarity, and they co-complex in mammalian cells. X-ray diffraction studies and homology modeling suggest that their N-terminal regions (residues 8-167) adopt continuous, extended α-helical coiled-coil structures, but no experimentally derived information on the structure of their C-terminal regions or the architecture of the full-length proteins is available. In the case of NDE1, no biophysical data exists. Here we characterize the structural architecture of both full-length proteins utilizing negative stain electron microscopy along with our established paradigm of chemical cross-linking followed by tryptic digestion, mass spectrometry, and database searching, which we enhance using isotope labeling for mixed NDE1-NDEL1. We determined that full-length NDE1 forms needle-like dimers and tetramers in solution, similar to crystal structures of NDEL1, as well as chain-like end-to-end polymers. The C-terminal domain of each protein, required for interaction with key protein partners dynein and DISC1 (disrupted-in-schizophrenia 1), includes a predicted disordered region that allows a bent back structure. This facilitates interaction of the C-terminal region with the N-terminal coiled-coil domain and is in agreement with previous results showing N- and C-terminal regions of NDEL1 and NDE1 cooperating in dynein interaction. It sheds light on recently identified mutations in the NDE1 gene that cause truncation of the encoded protein. Additionally, analysis of mixed NDE1-NDEL1 complexes demonstrates that NDE1 and NDEL1 can interact directly.

Structure and operation of the DNA-translocating type I DNA restriction enzymes.

Type I DNA restriction/modification (RM) enzymes are molecular machines found in the majority of bacterial species. Their early discovery paved the way for the development of genetic engineering. They control (restrict) the influx of foreign DNA via horizontal gene transfer into the bacterium while maintaining sequence-specific methylation (modification) of host DNA. The endonuclease reaction of these enzymes on unmethylated DNA is preceded by bidirectional translocation of thousands of base pairs of DNA toward the enzyme. We present the structures of two type I RM enzymes, EcoKI and EcoR124I, derived using electron microscopy (EM), small-angle scattering (neutron and X-ray), and detailed molecular modeling. DNA binding triggers a large contraction of the open form of the enzyme to a compact form. The path followed by DNA through the complexes is revealed by using a DNA mimic anti-restriction protein. The structures reveal an evolutionary link between type I RM enzymes and type II RM enzymes.

The structure of M.EcoKI Type I DNA methyltransferase with a DNA mimic antirestriction protein.

Type-I DNA restriction-modification (R/M) systems are important agents in limiting the transmission of mobile genetic elements responsible for spreading bacterial resistance to antibiotics. EcoKI, a Type I R/M enzyme from Escherichia coli, acts by methylation- and sequence-specific recognition, leading to either methylation of DNA or translocation and cutting at a random site, often hundreds of base pairs away. Consisting of one specificity subunit, two modification subunits, and two DNA translocase/endonuclease subunits, EcoKI is inhibited by the T7 phage antirestriction protein ocr, a DNA mimic. We present a 3D density map generated by negative-stain electron microscopy and single particle analysis of the central core of the restriction complex, the M.EcoKI M(2)S(1) methyltransferase, bound to ocr. We also present complete atomic models of M.EcoKI in complex with ocr and its cognate DNA giving a clear picture of the overall clamp-like operation of the enzyme. The model is consistent with a large body of experimental data on EcoKI published over 40 years.

Dodecameric structure of the small heat shock protein Acr1 from Mycobacterium tuberculosis.

Small heat shock proteins are a ubiquitous and diverse family of stress proteins that have in common an alpha-crystallin domain. Mycobacterium tuberculosis has two small heat shock proteins, Acr1 (alpha-crystallin-related protein 1, or Hsp16.3/16-kDa antigen) and Acr2 (HrpA), both of which are highly expressed under different stress conditions. Small heat shock proteins form large oligomeric assemblies and are commonly polydisperse. Nanoelectrospray mass spectrometry showed that Acr2 formed a range of oligomers composed of dimers and tetramers, whereas Acr1 was a dodecamer. Electron microscopy of Acr2 showed a variety of particle sizes. Using three-dimensional analysis of negative stain electron microscope images, we have shown that Acr1 forms a tetrahedral assembly with 12 polypeptide chains. The atomic structure of a related alpha-crystallin domain dimer was docked into the density to build a molecular structure of the dodecameric Acr1 complex. Along with the differential regulation of these two proteins, the differences in their quaternary structures demonstrated here supports their distinct functional roles.

Insight into the potential for DNA idiotypic fusion vaccines designed for patients by analysing xenogeneic anti-idiotypic antibody responses.

DNA vaccines induce immune responses against encoded proteins, and have clear potential for cancer vaccines. For B-cell tumours, idiotypic (Id) immunoglobulin encoded by the variable region genes provides a target antigen. When assembled as single chain Fv (scFv), and fused to an immunoenhancing sequence from tetanus toxin (TT), DNA fusion vaccines induce anti-Id antibodies. In lymphoma models, these antibodies have a critical role in mediating protection. For application to patients with lymphoma, two questions arise: first, whether pre-existing antibody against TT affects induction of anti-scFv antibodies; second, whether individual human scFv fusion sequences are able to fold consistently to generate antibodies able to recognize private conformational Id determinants expressed by tumour cells. Using xenogeneic vaccination with scFv sequences from four patients, we have shown that pre-existing anti-TT immunity slows, but does not prevent, anti-Id antibody responses. To determine folding, we have monitored the ability of nine DNAscFv-FrC patients' vaccines to induce xenogeneic anti-Id antibodies. Antibodies were induced in all cases, and were strikingly specific for each patient's immunoglobulin with little cross-reactivity between patients, even when similar VH or VL genes were involved. Blocking experiments with human serum confirmed reactivity against private determinants in 26-97% of total antibody. Both immunoglobulin G1 (IgG1) and IgG2a subclasses were present at 1.3 : 1-15 : 1 consistent with a T helper 2-dominated response. Xenogeneic vaccination provides a simple route for testing individual patients' DNAscFv-FrC fusion vaccines, and offers a strategy for production of anti-Id antibodies. The findings underpin the approach of DNA idiotypic fusion vaccination for patients with B-cell tumours.