Design of Compact Biomimetic Cellulose Binding Peptides as Carriers for Cellulose Catalytic Degradation.

The conversion of biomass into biofuels can reduce the strategic vulnerability of petroleum-based systems and at the same time have a positive effect on global climate issues. Lignocellulose is the cheapest and most abundant source of biomass and consequently has been widely considered as a source for liquid fuel. However, despite ongoing efforts, cellulosic biofuels are still far from commercial realization, one of the major bottlenecks being the hydrolysis of cellulose into simpler sugars. Inspired by the structural and functional modularity of cellulases used by many organisms for the breakdown of cellulose, we propose to mimic the cellulose binding domain (CBD) and the catalytic domain of these proteins by small molecular entities. Multiple copies of these mimics could subsequently be tethered together to enhance hydrolytic activity. In this work, we take the first step toward achieving this goal by applying computational approaches to the design of efficient, cost-effective mimetics of the CBD. The design is based on low molecular weight peptides that are amenable to large-scale production. We provide an optimized design of four short (i.e., ∼18 residues) peptide mimetics based on the three-dimensional structure of a known CBD and demonstrate that some of these peptides bind cellulose as well as or better than the full CBD. The structures of these peptides were studied by circular dichroism and their interactions with cellulose by solid phase NMR. Finally, we present a computational strategy for predicting CBD/peptide-cellulose binding free energies and demonstrate its ability to provide values in good agreement with experimental data. Using this computational model, we have also studied the dissociation pathway of the CBDs/peptides from the surface of cellulose.

REMD Simulations Reveal the Dynamic Profile and Mechanism of Action of Deleterious, Rescuing, and Stabilizing Perturbations to NBD1 from CFTR.

Cystic Fibrosis (CF) is a lethal, genetic disease caused by mutations to the CFTR chloride channel. The most common CF causing mutation is the deletion of F508 from the first Nucleotide Binding Domain (F508del-NBD1). This mutation leads to a thermally unstable domain and a misfolded, nonfunctioning CFTR. Replica Exchange MD simulations were used to simulate seven NBD1 constructs including wt and F508del-NBD1 both alone and in the presence of known rescuing mutations as well as F508del-NBD1 in complex with a known small (ligand) stabilizer. Analyzing the resulting trajectories suggests that differences in the biochemical properties of the constructs result from local and coupled differences in their dynamic profiles. A comparative analysis of these profiles as well as of the resulting trajectories reveals how the different perturbations exert their deleterious, rescuing, and stabilizing effects on NBD1. These simulations may therefore be useful for the design and mechanism-of-action analysis of new NBD1 stabilizers.

Vipegitide: a folded peptidomimetic partial antagonist of α2ß1 integrin with antiplatelet aggregation activity.

Linear peptides containing the sequence WKTSRTSHY were used as lead compounds to synthesize a novel peptidomimetic antagonist of α2ß1 integrin, with platelet aggregation-inhibiting activity, named Vipegitide. Vipegitide is a 13-amino acid, folded peptidomimetic molecule, containing two α-aminoisobutyric acid residues at positions 6 and 8 and not stable in human serum. Substitution of glycine and tryptophan residues at positions 1 and 2, respectively, with a unit of two polyethylene glycol (PEG) molecules yielded peptidomimetic Vipegitide-PEG2, stable in human serum for over 3 hours. Vipegitide and Vipegitide-PEG2 showed high potency (7×10(-10) M and 1.5×10(-10) M, respectively) and intermediate efficacy (40% and 35%, respectively) as well as selectivity toward α2 integrin in inhibition of adhesion of α1/α2 integrin overexpressing cells toward respective collagens. Interaction of both peptidomimetics with extracellular active domain of α2 integrin was confirmed in cell-free binding assay with recombinant α2 A-domain. Integrin α2ß1 receptor is found on the platelet membrane and triggers collagen-induced platelet aggregation. Vipegitide and Vipegitide-PEG2 inhibited α2ß1 integrin-mediated adhesion of human and murine platelets under the flow condition, by 50%. They efficiently blocked adenosine diphosphate- and collagen I-induced platelet aggregation in platelet rich plasma and whole human blood. Higher potency of Vipegitide than Vipegitide-PEG2 is consistent with results of computer modeling of the molecules in water. These peptidomimetic molecules were acutely tolerated in mice upon intravenous bolus injection of 50 mg/kg. These results underline the potency of Vipegitide and Vipegitide-PEG2 molecules as platelet aggregation-inhibiting drug lead compounds in antithrombotic therapy.

Vimocin and vidapin, cyclic KTS peptides, are dual antagonists of α1ß1/α2ß1 integrins with antiangiogenic activity.

Obtustatin and viperistatin, members of the disintegrin protein family, served as lead compounds for the synthesis of linear and cyclic peptides containing the KTS binding motif. The most active linear peptide, a viperistatin analog, indicated the importance of Cys(19) and Cys(29), as well as the presence of Arg at position 24 for their biologic activity, and was used as the basic sequence for the synthesis of cyclic peptides. Vimocin (compound 6) and vidapin (compound 10) showed a high potency (IC50 = 0.17 nM) and intermediate efficacy (20 and 40%) in inhibition of adhesion of α1/α2 integrin overexpressor cells to respective collagens. Vimocin was more active in inhibition of the wound healing (53%) and corneal micropocket (17%) vascularization, whereas vidapin was more potent in inhibition of migration in the Matrigel tube formation assay (90%). Both compounds similarly inhibited proliferation (50-90%) of endothelial cells, and angiogenesis induced by vascular endothelial growth factor (80%) and glioma (55%) in the chorioallantoic membrane assay. These peptides were not toxic to endothelial cell cultures and caused no acute toxicity upon intravenous injection in mice, and were stable for 10-30 hours in human serum. The in vitro and in vivo potency of the peptides are consistent with conformational ensembles and "bioactive" space shared by obtustatin and viperistatin. These findings suggest that vimocin and vidapin can serve as dual α1ß1/α2ß1 integrin antagonists in antiangiogenesis and cancer therapy.

Conformational changes relevant to channel activity and folding within the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator.

Deletion of Phe-508 (F508del) in the first nucleotide binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to defects in folding and channel gating. NMR data on human F508del NBD1 indicate that an H620Q mutant, shown to increase channel open probability, and the dual corrector/potentiator CFFT-001 similarly disrupt interactions between ß-strands S3, S9, and S10 and the C-terminal helices H8 and H9, shifting a preexisting conformational equilibrium from helix to coil. CFFT-001 appears to interact with ß-strands S3/S9/S10, consistent with docking simulations. Decreases in T(m) from differential scanning calorimetry with H620Q or CFFT-001 suggest direct compound binding to a less thermostable state of NBD1. We hypothesize that, in full-length CFTR, shifting the conformational equilibrium to reduce H8/H9 interactions with the uniquely conserved strands S9/S10 facilitates release of the regulatory region from the NBD dimerization interface to promote dimerization and thereby increase channel open probability. These studies enabled by our NMR assignments for F508del NBD1 provide a window into the conformational fluctuations within CFTR that may regulate function and contribute to folding energetics.

Flexible protein-protein docking based on Best-First search algorithm.

We developed a new high resolution protein-protein docking method based on Best-First search algorithm that loosely imitates protein-protein associations. The method operates in two stages: first, we perform a rigid search on the unbound proteins. Second, we search alternately on rigid and flexible degrees of freedom starting from multiple configurations from the rigid search. Both stages use heuristics added to the energy function, which causes the proteins to rapidly approach each other and remain adjacent, while optimizing on the energy. The method deals with backbone flexibility explicitly by searching over ensembles of conformations generated before docking. We ran the rigid docking stage on 66 complexes and grouped the results into four classes according to evaluation criteria used in Critical Assessment of Predicted Interactions (CAPRI; "high," "medium," "acceptable," and "incorrect"). Our method found medium binding conformations for 26% of the complexes and acceptable for additional 44% among the top 10 configurations. Considering all the configurations, we found medium binding conformations for 55% of the complexes and acceptable for additional 39% of the complexes. Introducing side-chains flexibility in the second stage improves the best found binding conformation but harms the ranking. However, introducing side-chains and backbone flexibility improve both the best found binding conformation and the best found conformation in the top 10. Our approach is a basis for incorporating multiple flexible motions into protein-protein docking and is of interest even with the current use of a simple energy function.

Constructing ensembles of flexible fragments in native proteins by iterative stochastic elimination is relevant to protein-protein interfaces.

We investigate the extent to which ensembles of flexible fragments (FF), generated by our loop conformational search method, include conformations that are near experimental and reflect conformational changes that these FFs undergo when binary protein-protein complexes are formed. Twenty-eight FFs, which are located in protein-protein interfaces and have different conformations in the bound structure (BS) and unbound structure (UbS) were extracted. The conformational space of these fragments in the BS and UbS was explored with our method which is based on the iterative stochastic elimination (ISE) algorithm. Conformational search of BSs generated bound ensembles and conformational search of UbSs produced unbound ensembles. ISE samples conformations near experimental (less than 1.05 A root mean square deviation, RMSD) for 51 out of the 56 examined fragments in the bound and unbound ensembles. In 14 out of the 28 unbound fragments, it also samples conformations within 1.05 A from the BS in the unbound ensemble. Sampling the bound conformation in the unbound ensemble demonstrates the potential biological relevance of the predicted ensemble. The 10 lowest energy conformations are the best choice for docking experiments, compared with any other 10 conformations of the ensembles. We conclude that generating conformational ensembles for FFs with ISE is relevant to FF conformations in the UbS and BS. Forming ensembles of the isolated proteins with our method prior to docking represents more comprehensively their inherent flexibility and is expected to improve docking experiments compared with results obtained by docking only UbSs.