Validation of the GROMOS force-field parameter set 45A3 against nuclear magnetic resonance data of hen egg lysozyme

The quality of molecular dynamics (MD) simulations of proteins depends critically on the biomolecular force field that is used. Such force fields are defined by force-field parameter sets, which are generally determined and improved through calibration of properties of small molecules against experimental or theoretical data. By application to large molecules such as proteins, a new force-field parameter set can be validated. We report two 3.5ns molecular dynamics simulations of hen egg white lysozyme in water applying the widely used GROMOS force-field parameter set 43A1 and a new set 45A3. The two MD ensembles are evaluated against NMR spectroscopic data NOE atom-atom distance bounds, 3JNH α and 3Jαβ coupling constants, and 15N relaxation data. It is shown that the two sets reproduce structural properties about equally well. The 45A3 ensemble fulfills the atom-atom distance bounds derived from NMR spectroscopy slightly less well than the 43A1 ensemble, with most of the NOE distance violations in both ensembles involving residues located in loops or flexible regions of the protein. Convergence patterns are very similar in both simulations atom-positional root-mean-square differences (RMSD) with respect to the X-ray and NMR model structures and NOE inter-proton distances converge within 1.0-1.5ns while backbone 3JHNα-coupling constants and 1H- 15N order parameters take slightly longer, 1.0-2.0ns. As expected, side-chain 3Jαβ-coupling constants and 1H- 15N order parameters do not reach full convergence for all residues in the time period simulated. This is particularly noticeable for side chains which display rare structural transitions. When comparing each simulation trajectory with an older and a newer set of experimental NOE data on lysozyme, it is found that the newer, larger, set of experimental data agrees as well with each of the simulations. In other words, the experimental data converged towards the theoretical resul

AGGRESCAN: a server for the prediction and evaluation of "hot spots" of aggregation in polypeptides

BACKGROUND: Protein aggregation correlates with the development of several debilitating human disorders of growing incidence, such as Alzheimer's and Parkinson's diseases. On the biotechnological side, protein production is often hampered by the accumulation of recombinant proteins into aggregates. Thus, the development of methods to anticipate the aggregation properties of polypeptides is receiving increasing attention. AGGRESCAN is a web-based software for the prediction of aggregation-prone segments in protein sequences, the analysis of the effect of mutations on protein aggregation propensities and the comparison of the aggregation properties of different proteins or protein sets. RESULTS: AGGRESCAN is based on an aggregation-propensity scale for natural amino acids derived from in vivo experiments and on the assumption that short and specific sequence stretches modulate protein aggregation. The algorithm is shown to identify a series of protein fragments involved in the aggregation of disease-related proteins and to predict the effect of genetic mutations on their deposition propensities. It also provides new insights into the differential aggregation properties displayed by globular proteins, natively unfolded polypeptides, amyloidogenic proteins and proteins found in bacterial inclusion bodies. CONCLUSION: By identifying aggregation-prone segments in proteins, AGGRESCAN shall facilitate (i) the identification of possible therapeutic targets for anti-depositional strategies in conformational diseases and (ii) the anticipation of aggregation phenomena during storage or recombinant production of bioactive polypeptides or polypeptide sets

Structural and biochemical characterization of NarE, an iron-containing ADP-ribosyltransferase from Neisseria meningitidis.

Abstract NarE is a 16 kDa protein identified from Neisseria meningitidis, one of the bacterial pathogens responsible for meningitis. NarE belongs to the family of ADP-ribosyltransferases (ADPRT) and catalyzes the transfer of ADP-ribose moieties to arginine residues in target protein acceptors. Many pathogenic bacteria utilize ADP-ribosylating toxins to modify and alter essential functions of eukaryotic cells. NarE is further the first ADPRT which could be shown to bind iron through a Fe-S center, which is crucial for the catalytic activity. Here we present the NMR solution structure of NarE, which shows structural homology to other ADPRTs. Using NMR titration experiments we could identify from Chemical Shift Perturbation data both the NAD binding site, which is in perfect agreement with a consensus sequence analysis between different ADPRTs, as well as the iron coordination site, which consists of 2 cysteines and 2 histidines. This atypical iron coordination is also capable to bind zinc. These results could be fortified by site-directed mutagenesis of the catalytic region, which identified two functionally crucial residues. We could further identify a main interaction region of NarE with antibodies using two complementary methods based on antibody immobilization, proteolytic digestion, and mass spectrometry. This study combines structural and functional features of NarE providing for the first time a characterization of an iron-dependent ADPRT

On the transferability of the SPCAL water model to biomolecular simulation

We investigated the performance of the recently developed SPC/L model for liquid water, as a pure liquid, in binary mixtures with DMSO, and as a solvent model in a peptide folding simulation. Additionally, in order to test the compatibility with the GROMOS biomolecular force field, free energies of hydration of a set of representative compounds were computed. The results are compared to those for the well established SPC water model, which is generally used as a solvent model in conjunction with the GROMOS force field already for more than two decades. It turns out that as a pure liquid and in binary mixtures with DMSO the SPC/L model outperforms SPC, whereas as solvent in combination with the GROMOS force field both models perform equally well

A potential new, stable state of the E-cadherin strand-swapped dimer in solution

E-cadherin is a transmembrane glycoprotein that facilitates inter-cellular adhesion in the epithelium. The ectodomain of the native structure is comprised of five repeated immunoglobulin-like domains. All E-cadherin crystal structures show the protein in one of three alternative conformations: a monomer, a strand-swapped trans homodimer and the so-called X-dimer, which is proposed to be a kinetic intermediate to forming the strand-swapped trans homodimer. However, previous studies have indicated that even once the trans strand-swapped dimer is formed, the complex is highly dynamic and the E-cadherin monomers may reorient relative to each other. Here, molecular dynamics simulations have been used to investigate the stability and conformational flexibility of the human E-cadherin trans strand-swapped dimer. In four independent, 100 ns simulations, the dimer moved away from the starting structure and converged to a previously unreported structure, which we call the Y-dimer. The Y-dimer was present for over 90% of the combined simulation time, suggesting that it represents a stable conformation of the E-cadherin dimer in solution. The Y-dimer conformation is stabilised by interactions present in both the trans strand-swapped dimer and X-dimer crystal structures, as well as additional interactions not found in any E-cadherin dimer crystal structures. The Y-dimer represents a previously unreported, stable conformation of the human E-cadherin trans strand-swapped dimer and suggests that the available crystal structures do not fully capture the conformations that the human E-cadherin trans homodimer adopts in solution

Spain: Underwater Exploration on a Narrow Continental Shelf

In spite of Spain’s long coastline (nearly 8000 km) and its well-established tradition in underwater archaeology, the prehistoric settlement of the continental shelf is practically unknown with very few finds. Underwater research has focused on naval archaeology and, until very recently, no attempt had been made to look for prehistoric underwater sites. In the past decade,new research projects have been launched to explore selected areas on the Cantabrian shelf and offshore of Gibraltar. This chapter summarises the currently available evidence of submerged prehistoric archaeology and the preliminary results of these new project

Mechanism of 150-cavity formation in influenza neuraminidase

The recently discovered 150-cavity in the active site of group-1 influenza A neuraminidase (NA) proteins provides a target for rational structure-based drug development to counter the increasing frequency of antiviral resistance in influenza. Surprisingly, the 2009 H1N1 pandemic virus (09N1) neuramidase was crystalized without the 150-cavity characteristic of group-1 NAs. Here we demonstrate, through a total sum of 1.6 μs of biophysical simulations, that 09N1 NA exists in solution preferentially with an open 150-cavity. Comparison with simulations using avian N1, human N2 and 09N1 with a I149V mutation and an extensive bioinformatics analysis suggests that the conservation of a key salt bridge is crucial in the stabilization of the 150-cavity across both subtypes. This result provides an atomic-level structural understanding of the recent finding that antiviral compounds designed to take advantage of contacts in the 150-cavity can inactivate both 2009 H1N1 pandemic and avian H5N1 viruses

BPSL1626 : Reverse and Structural Vaccinology Reveal a Novel Candidate for Vaccine Design Against Burkholderia pseudomallei

Due to significant advances in computational biology, protein prediction, together with antigen and epitope design, have rapidly moved from conventional methods, based on experimental approaches, to in silico-based bioinformatics methods. In this context, we report a reverse vaccinology study that identified a panel of 104 candidate antigens from the Gram-negative bacterial pathogen Burkholderia pseudomallei, which is responsible for the disease melioidosis. B. pseudomallei can cause fatal sepsis in endemic populations in the tropical regions of the world and treatment with antibiotics is mostly ineffective. With the aim of identifying potential vaccine candidates, we report the experimental validation of predicted antigen and type I fimbrial subunit, BPSL1626, which we show is able to recognize and bind human antibodies from the sera of Burkholderia infected patients and to stimulate T-lymphocytes in vitro. The prerequisite for a melioidosis vaccine, in fact, is that both antibody- and cell-mediated immune responses must be triggered. In order to reveal potential antigenic regions of the protein that may aid immunogen re-design, we also report the crystal structure of BPSL1626 at 1.9 angstrom resolution on which structure-based epitope predictions were based. Overall, our data suggest that BPSL1626 and three epitope regions here-identified can represent viable candidates as potential antigenic molecules

Molecular dynamics simulation of biomolecular systems

The group for computer-aided chemistry at the ETH Zurich focuses its research on the development of methodology to simulate the behavior of biomolecular systems and the use of simulation techniques to analyze and understand biomolecular processes at the atomic level. Here, the current research directions are briefly reviewed and illustrated with a few examples