Protein flexibility and conformational states of Leishmania antigen eIF-4A: identification of a novel plausible protein adjuvant using comparative genomics and molecular modeling.

Recent homology modeling studies have identified specific residues (epitope) of the Leishmania RNA helicase protein (LmeIF) that stimulates production of IL-12 cytokine. However, question remains concerning how LmeIF's N-terminal moiety initiates adjuvant effects. Extensive molecular modeling combining the normal mode analysis (NMA) and molecular dynamics simulations, in the present study, has demonstrated that the LmeIF structure may exist in two different forms corresponding to the extended and collapsed (closed) states of the entire structure. The computational results showed that the two domains of the LmeIF structure tend to undergo large fluctuations in a concerted fashion and have strong effect on the solvent accessible surface of the epitope situated on the N-terminal structure. The conformational freedom of the C-terminal domains may explain why the entire LmeIF protein is not as active as the N-terminal moiety. Thereafter, a comparative genome analysis with subsequent homology modeling and molecular electrostatic potential (MEP) techniques allowed us to predict a novel and plausible RNA helicase (LI-helicase) from the Listeria source with adjuvant property as observed for the Leishmania eIF-4A protein. The structural folding and MEP maps revealed similar topologies of the epitope of both LmeIF and LI-helicase proteins and striking identity in the local disposition of the charged groups. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:7.

Unveiling the unfolding pathway of F5F8D disorder-associated D81H/V100D mutant of MCFD2 via multiple molecular dynamics simulations.

Combined factor deficiency (F5F8D) is a rare autosomal recessive disorder caused by mutations in the LMAN1 or MCFD2 genes. It has been proposed that this pathogenic process occurs via a multi-step pathway involving metal loss, EF-hand-Ca21 dissociation and assembly of misfolded MCFD2-LMAN1 complex. Here, we have investigated the solution conformations of the MCFD2((D81H,V100D)) protein mutant through extensive molecular dynamics (MD) simulations. The V100D, one of the many MCFD2 mutations known to be associated to F5F8D, is difficult to be reconciled with the pathway model because it is located far from the metal sites and the MCFD2/LMAN1 interface. Consequently, an inspection of all the steps involved in D81H/V100D MCFD2 misfolding is expected to provide hints in the understanding of the molecular basis of the disease. A comparison with parallel studies carried out for the Wild-Type (WT) MCFD2 pointed out that the mutation decreases the affinity of the protein for the Ca21 ion. Multiple explicit solvents MD simulations (50 ns) performed on the two proteins revealed that in the WT protein, stable H-bond network and compact hydrophobic core region are created thus confirming a pivotal role of this region in driving the biophysical properties of the entire protein. In fact it is shown that the V100D mutation, although located far away the EF-hand domain, may induce subtle modification in the structural core of MCFD2 leading to the loosening of metal binding and to the formation of metastable intermediate states along the unfolding pathway. The native-like hydrophobic cluster formed near the V100 residue in the wild-type protein is disrupted by the negatively charged Asparagine residue. Furthermore, the presence of the D81H mutation in the EF-1 hand domain may also increase the protein unfolding rate and consequently prevent the formation of the MCFD2-LMAN1 complex. The detailed structural insights obtained from our large-scale simulations complement the clinical features and offer useful insights into the mechanism behind MCFD2 protein misfolding.