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 M2S1 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.

Small heat shock proteins are a ubiquitous and diverse family of stress proteins that have in common an ␣-crystallin domain. Mycobacterium tuberculosis has two small heat shock proteins, Acr1 (␣-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 ␣-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.Tuberculosis is one of the major killer infective diseases and represents a threat in both underdeveloped and developed countries due both to increased drug resistance and to the high levels of TB occurrence in human immunodeficiency virus-infected individuals. About one-third of the world's population is infected with the bacterium, which can spend many years in a dormant state inside lung granulomas. Disease is often the result of the bacteria sequestered inside lung macrophages being activated when the immune system of the infected individual is weakened.The ␣-crystallin/small heat shock protein (sHSP) 2 family is ubiquitous throughout nature and carries out a general cellular protective role in preventing aggregation of denatured proteins and facilitating subsequent refolding by other chaperones (1, 2). This function is particularly important in Mycobacterium tuberculosis, which must be able to survive an inhospitable environment while sequestered within phagosomes of alveolar macrophages. sHSPs typically form large homo-oligomeric complexes and often exhibit a high degree of dynamic subunit exchange, which is thought to contribute to, or be a consequence of, their chaperone function (1, 3).Acr1 3 (also known as Hsp16.3/HspX/16-kDa Antigen/Rv2031c) is a 16.3-kDa protein, one of two members of the sHSP family found in M. tuberculosis. Acr2 (also known as Heat-stress-induced Ribosomeassociated Protein A/HrpA/Rv0251c), the second sHSP, has a mass of 17.8 kDa (4,5). Both proteins, like all sHSPs, share a conserved central domain of ϳ90 amino acids called the ␣-crystallin domain and have divergent N-and C-terminal extensions. The sequence similarity between Acr1 and Acr2 is 43% overall and 55% in the crystallin domain. Free growing Mycobacteria (M. smegmatis and M. marinarium) have homologues of both Acr1 and Acr2 and a third Acr protein, Acr3, which is most similar to the single sHSP in M. leprae (6).Acr1 is the most...

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.

Background: NDE1 and NDEL1 are neurodevelopmental and mitotic proteins with extended coiled-coil N termini, but unknown C-terminal structure.Results: Recombinant NDE1/NDEL1 form dimers and tetramers in which their C termini interact with their N-terminal domains.Conclusion: NDE1/NDEL1 each adopt a sharply bent back structure.Significance: This explains the existence of two distinct dynein-binding domains on NDE1/NDEL1 and instability of disease-associated mutants lacking C termini.

SUMMARYDNA 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 V H or V L 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.