A candidate ion-retaining state in the inward-facing conformation of sodium/galactose symporter: Clues from atomistic simulations

The recent Vibrio parahaemolyticus sodium/galactose (vSGLT) symporter crystal structure captures the protein in an inward-facing substrate-bound conformation, with the sodium ion placed, by structural alignment, in a site equivalent to the Na2 site of the leucine transporter (LeuT). A recent study, based on molecular dynamics simulations, showed that the sodium ion spontaneously leaves its initial position diffusing outside vSGLT, toward the intracellular space. This suggested that the crystal structure corresponds to an ion-releasing state of the transporter. Here, using metadynamics, we identified a more stable Na+ binding site corresponding to a putative ion-retaining state of the transporter. In addition, our simulations, consistently with mutagenesis studies, highlight the importance of D189 that, without being one of the NA(+)-coordinating residues, regulates its binding/release

In Vitro Evolution of Allergy Vaccine Candidates, with Maintained Structure, but Reduced B Cell and T Cell Activation Capacity

Allergy and asthma to cat (Felis domesticus) affects about 10% of the population in affluent countries. Immediate allergic symptoms are primarily mediated via IgE antibodies binding to B cell epitopes, whereas late phase inflammatory reactions are mediated via activated T cell recognition of allergen-specific T cell epitopes. Allergen-specific immunotherapy relieves symptoms and is the only treatment inducing a long-lasting protection by induction of protective immune responses. The aim of this study was to produce an allergy vaccine designed with the combined features of attenuated T cell activation, reduced anaphylactic properties, retained molecular integrity and induction of efficient IgE blocking IgG antibodies for safer and efficacious treatment of patients with allergy and asthma to cat. The template gene coding for rFel d 1 was used to introduce random mutations, which was subsequently expressed in large phage libraries. Despite accumulated mutations by up to 7 rounds of iterative error-prone PCR and biopanning, surface topology and structure was essentially maintained using IgE-antibodies from cat allergic patients for phage enrichment. Four candidates were isolated, displaying similar or lower IgE binding, reduced anaphylactic activity as measured by their capacity to induce basophil degranulation and, importantly, a significantly lower T cell reactivity in lymphoproliferative assays compared to the original rFel d 1. In addition, all mutants showed ability to induce blocking antibodies in immunized mice.The approach presented here provides a straightforward procedure to generate a novel type of allergy vaccines for safer and efficacious treatment of allergic patients

Drug export and allosteric coupling in a multidrug transporter revealed by molecular simulations

Multidrug resistance is a serious problem in current chemotherapy. The efflux system largely responsible for resistance in Escherichia coli contains the drug transporter, AcrB. The structures of AcrB were solved in 2002 as the symmetric homo-trimer, and then in 2006 as the asymmetric homo-trimer. The latter suggested a functionally rotating mechanism. Here, by molecular simulations of the AcrB porter domain, we uncovered allosteric coupling and the drug export mechanism in the AcrB trimer. Allosteric coupling stabilized the asymmetric structure with one drug molecule bound, which validated the modelling. Drug dissociation caused a conformational change and stabilized the symmetric structure, providing a unified view of the structures reported in 2002 and 2006. A dynamic study suggested that, among the three potential driving processes, only protonation of the drug-bound protomer can drive the functional rotation and simultaneously export the drug

Three dimensional structure directs T-cell epitope dominance associated with allergy

<p>Abstract</p> <p>Background</p> <p>CD4+ T-cell epitope immunodominance is not adequately explained by peptide selectivity in class II major histocompatibility proteins, but it has been correlated with adjacent segments of conformational flexibility in several antigens.</p> <p>Methods</p> <p>The published T-cell responses to two venom allergens and two aeroallergens were used to construct profiles of epitope dominance, which were correlated with the distribution of conformational flexibility, as measured by crystallographic B factors, solvent-accessible surface, COREX residue stability, and sequence entropy.</p> <p>Results</p> <p>Epitopes associated with allergy tended to be excluded from and lie adjacent to flexible segments of the allergen.</p> <p>Conclusion</p> <p>During the initiation of allergy, the N- and/or C-terminal ends of proteolytic processing intermediates were preferentially loaded into antigen presenting proteins for the priming of CD4+ T cells.</p

Investigating Homology between Proteins using Energetic Profiles

Accumulated experimental observations demonstrate that protein stability is often preserved upon conservative point mutation. In contrast, less is known about the effects of large sequence or structure changes on the stability of a particular fold. Almost completely unknown is the degree to which stability of different regions of a protein is generally preserved throughout evolution. In this work, these questions are addressed through thermodynamic analysis of a large representative sample of protein fold space based on remote, yet accepted, homology. More than 3,000 proteins were computationally analyzed using the structural-thermodynamic algorithm COREX/BEST. Estimated position-specific stability (i.e., local Gibbs free energy of folding) and its component enthalpy and entropy were quantitatively compared between all proteins in the sample according to all-vs.-all pairwise structural alignment. It was discovered that the local stabilities of homologous pairs were significantly more correlated than those of non-homologous pairs, indicating that local stability was indeed generally conserved throughout evolution. However, the position-specific enthalpy and entropy underlying stability were less correlated, suggesting that the overall regional stability of a protein was more important than the thermodynamic mechanism utilized to achieve that stability. Finally, two different types of statistically exceptional evolutionary structure-thermodynamic relationships were noted. First, many homologous proteins contained regions of similar thermodynamics despite localized structure change, suggesting a thermodynamic mechanism enabling evolutionary fold change. Second, some homologous proteins with extremely similar structures nonetheless exhibited different local stabilities, a phenomenon previously observed experimentally in this laboratory. These two observations, in conjunction with the principal conclusion that homologous proteins generally conserved local stability, may provide guidance for a future thermodynamically informed classification of protein homology

The bacterial dicarboxylate transporter VcINDY uses a two-domain elevator-type mechanism

Secondary transporters use alternating-access mechanisms to couple uphill substrate movement to downhill ion flux. Most known transporters use a 'rocking bundle' motion, wherein the protein moves around an immobile substrate-binding site. However, the glutamate-transporter homolog GltPh translocates its substrate-binding site vertically across the membrane, through an 'elevator' mechanism. Here, we used the 'repeat swap' approach to computationally predict the outward-facing state of the Na(+)/succinate transporter VcINDY, from Vibrio cholerae. Our model predicts a substantial elevator-like movement of VcINDY's substrate-binding site, with a vertical translation of ~15 Å and a rotation of ~43°. Our observation that multiple disulfide cross-links completely inhibit transport provides experimental confirmation of the model and demonstrates that such movement is essential. In contrast, cross-links across the VcINDY dimer interface preserve transport, thus revealing an absence of large-scale coupling between protomers

Unusual segmental flexibility in a region of tobacco mosaic virus coat protein

ASSEMBLY of tobacco mosaic virus (TMV) is known to occur by threading in the RNA on the inner side of the growing hollow rodlet of viral coat protein, and is thought to involve a pre-assembled double disk as an intermediate1–3. Given the structure of the subunit4, such a mechanism requires that it be possible to move the segment of the polypeptide chain which separates the nucleic acid groove from the lumen of the cylinder so that it is out of the way in the assembly process. Evidence suggesting that the segment in question has some flexibility comes from X-ray diffraction studies of the virus and its components. In the intact virus studied by the Heidelberg group this segment (residues 102–112) apparently exists largely as an a-helix (the V-helix)4; on the other hand, in the crystals of the isolated coat protein disk studied by the Cambridge group, residues 102–112 and adjacent segments (88–100, 112–114) do not give a well-defined electron density, reflecting a high degree of disorder5. However, X-ray evidence is inconclusive on this point, as it does not distinguish between thermal disorder indicating flexibility and static disorder indicating irregular packing of the segment in the crystal, de Wit et al.6,7 have pointed out that there must be internal mobility in the TMV coat protein, based on the results of 13C NMR of various TMV protein aggregates. Unfortunately, their results do not show whether or not there is flexibility in a particular segment of the protein. In an effort to distinguish between the thermal disorder and the static disorder in the V-helix segment, we describe here our investigation of the problem by high-resolution proton nuclear magnetic resonance (NMR) studies, and report a flexibility due to thermal motional disorder