De novo backbone scaffolds for protein design

In recent years, there have been significant advances in the field of computational protein design including the successful computational design of enzymes based on backbone scaffolds from experimentally solved structures. It is likely that large‐scale sampling of protein backbone conformations will become necessary as further progress is made on more complicated systems. Removing the constraint of having to use scaffolds based on known protein backbones is a potential method of solving the problem. With this application in mind, we describe a method to systematically construct a large number of de novo backbone structures from idealized topological forms in a top–down hierarchical approach. The structural properties of these novel backbone scaffolds were analyzed and compared with a set of high‐resolution experimental structures from the protein data bank (PDB). It was found that the Ramachandran plot distribution and relative γ‐ and β‐turn frequencies were similar to those found in the PDB. The de novo scaffolds were sequence designed with RosettaDesign, and the energy distributions and amino acid compositions were comparable with the results for redesigned experimentally solved backbones. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Computational backbone design enables soluble engineering of transferrin receptor apical domain

Supply of iron into human cells is achieved by iron carrier protein transferrin and its receptor that upon complex formation get internalized by endocytosis. Similarly, the iron needs to be delivered into the brain, and necessitates the transport across the blood-brain barrier. While there are still unanswered questions about these mechanisms, extensive efforts have been made to use the system for delivery of therapeutics into biological compartments. The dimeric form of the receptor, where each subunit consists of three domains, further complicates the detailed investigation of molecular determinants responsible for guiding the receptor interactions with other proteins. Especially the apical domain's biological function has been elusive. To further the study of transferrin receptor, we have computationally decoupled the apical domain for soluble expression, and validated the design strategy by structure determination. Besides presenting a methodology for solubilizing domains, the results will allow for study of apical domain's function.     

Computational protein design using flexible backbone remodeling and resurfacing: case studies in structure-based antigen design

Computational protein design has promise for vaccine design and other applications. We previously transplanted the HIV 4E10 epitope onto non-HIV protein scaffolds for structural stabilization and immune presentation. Here, we developed two methods to optimize the structure of an antigen, flexible backbone remodeling and resurfacing, and we applied these methods to a 4E10 scaffold. In flexible-backbone remodeling, an existing backbone segment is replaced by a de novo designed segment of prespecified length and secondary structure. With remodeling, we replaced a potentially immunodominant domain on the scaffold with a helix-loop segment that made intimate contact to the protein core. All three domain trim designs tested experimentally had improved thermal stability and similar binding affinity for the 4E10 antibody compared to the parent scaffold. A crystal structure of one design had a 0.8 Å backbone RMSD to the computational model in the rebuilt region. Comparison of parent and trimmed scaffold reactivity to anti-parent sera confirmed the deletion of an immunodominant domain. In resurfacing, the surface of an antigen outside a target epitope is redesigned to obtain variants that maintain only the target epitope. Resurfaced variants of two scaffolds were designed in which 50 positions amounting to 40% of the protein sequences were mutated. Surface-patch analyses indicated that most potential antibody footprints outside the 4E10 epitope were altered. The resurfaced variants maintained thermal stability and binding affinity. These results indicate that flexible-backbone remodeling and resurfacing are useful tools for antigen optimization and protein engineering generally.     

Dipeptide backbone conformation and antibody recognition of a viral octapeptide epitope.

The peptide YKGTMDSG (Tyr-Lys-Gly-Thr-Met-Asp-Ser-Gly) represents an important antigenic determinant from the glycoprotein G2 of the pathogenic Rift Valley fever virus. By preparing a series of single-residue substitution peptides, the importance to antigenicity of individual residues within this octapeptide has been determined. Here, we investigated a simple and rapid computational analysis to test for correlations between the observed antigenicity of the substitution analogue peptides and the calculated conformational preferences in local regions of the peptides. Conformational energy analyses were carried out on all dipeptide combinations represented in the wild-type octapeptide and in the single-residue substitution analogue peptides. Conformational similarities and differences between wild-type and substitution dipeptide pairs were determined. The results of these computational analyses were then compared with the data on the relative antigenicity of the wild-type octapeptide and the substitution analogues. This comparison revealed a positive correlation. Substitution peptides showing changes in antigenicity possessed significant changes in the calculated backbone conformation relative to wild type in the dipeptides encompassing the residue substitution. Substitution peptides showing no change in antigenicity similarly showed no significant changes in dipeptide conformation. The potential utility of dipeptide conformational energy analyses and this preliminary structure-activity correlation are discussed.     

T-cell epitope vaccine design by immunoinformatics

Vaccination is generally considered to be the most effective method of preventing infectious diseases. All vaccinations work by presenting a foreign antigen to the immune system in order to evoke an immune response. The active agent of a vaccine may be intact but inactivated (‘attenuated’) forms of the causative pathogens (bacteria or viruses), or purified components of the pathogen that have been found to be highly immunogenic. The increased understanding of antigen recognition at molecular level has resulted in the development of rationally designed peptide vaccines. The concept of peptide vaccines is based on identification and chemical synthesis of B-cell and T-cell epitopes which are immunodominant and can induce specific immune responses. The accelerating growth of bioinformatics techniques and applications along with the substantial amount of experimental data has given rise to a new field, called immunoinformatics. Immunoinformatics is a branch of bioinformatics dealing with in silico analysis and modelling of immunological data and problems. Different sequence- and structure-based immunoinformatics methods are reviewed in the paper.     

Towards echinomycin mimetics by grafting quinoxaline residues on glycophane scaffolds

Echinomycin is a natural depsipeptide, which is a bisintercalator, inserting quinoxaline units preferentially adjacent to CG base pairs of DNA. Herein the design and synthesis of echinomycin mimetics based on grafting of two quinoxaline residues onto a macrocyclic scaffold (glycophane) is addressed. Binding of the compounds to calf-thymus DNA was studied using UV-vis and steady state fluorescence spectroscopy, as well as thermal denaturation. An interesting observation was enhancement of fluorescence emission for the peptidomimetics on binding to DNA, which contrasted with observations for echinomycin. Molecular dynamics simulations were exploited to explore in more detail if bis-intercalation to DNA was possible for one of the glycophanes. Bis-intercalating echinomycin complexes with DNA were found to be stable during 20ns simulations at 298K. However, the MD simulations of a glycophane complexed with a DNA octamer displayed very different behaviour to echinomycin and its quinoxaline units were found to rapidly migrate out from the intercalation site. Release of bis-intercalation strain occurred with only one of the quinoxaline chromophores remaining intercalated throughout the simulation. The distance between the quinoxaline residues in the glycophane at the end of the MD simulation was 7.3-7.5?, whereas in echinomycin, the distance between the residues was ～11?, suggesting that longer glycophane scaffolds would be required to generate bis-intercalating echinomycin mimetics.     

Computational assignment of protein backbone NMR peaks by efficient bounding and filtering

NMR resonance assignment is one of the key steps in solving an NMR protein structure. The assignment process links resonance peaks to individual residues of the target protein sequence, providing the prerequisite for establishing intra- and inter-residue spatial relationships between atoms. The assignment process is tedious and time-consuming, which could take many weeks. Though there exist a number of computer programs to assist the assignment process, many NMR labs are still doing the assignments manually to ensure quality. This paper presents a new computational method based on the combination of a suite of algorithms for automating the assignment process, particularly the process of backbone resonance peak assignment. We formulate the assignment problem as a constrained weighted bipartite matching problem. While the problem, in the most general situation, is NP-hard, we present an efficient solution based on a branch-and-bound algorithm with effective bounding techniques using two recently introduced approximation algorithms. We also devise a greedy filtering algorithm for reducing the search space. Our experimental results on 70 instances of (pseudo) real NMR data derived from 14 proteins demonstrate that the new solution runs much faster than a recently introduced (exhaustive) two-layer algorithm and recovers more correct peak assignments than the two-layer algorithm. Our result demonstrates that integrating different algorithms can achieve a good tradeoff between backbone assignment accuracy and computation time.     

Enzyme design by chemical modification of protein scaffolds

Covalent modification methods allow an almost unlimited range of functionality to be introduced into proteins. In concert with genetic techniques, chemical strategies have had significant impact in the field of enzyme design. Major recent developments include introducing catalytic activity into inactive proteins, modifying the selectivity and/or reactivity of existing enzymes and designing novel enzyme-based biosensors. New chemical methods promise to further increase the range of functionality that can be incorporated into proteins. These results suggest that semi-synthetic methods will play a key role in the development of future biocatalysts.     

Computational epitope map of SARS-CoV-2 spike protein

The primary immunological target of COVID-19 vaccines is the SARS-CoV-2 spike (S) protein. S is exposed on the viral surface and mediates viral entry into the host cell. To identify possible antibody binding sites, we performed multi-microsecond molecular dynamics simulations of a 4.1 million atom system containing a patch of viral membrane with four full-length, fully glycosylated and palmitoylated S proteins. By mapping steric accessibility, structural rigidity, sequence conservation, and generic antibody binding signatures, we recover known epitopes on S and reveal promising epitope candidates for structure-based vaccine design. We find that the extensive and inherently flexible glycan coat shields a surface area larger than expected from static structures, highlighting the importance of structural dynamics. The protective glycan shield and the high flexibility of its hinges give the stalk overall low epitope scores. Our computational epitope-mapping procedure is general and should thus prove useful for other viral envelope proteins whose structures have been characterized.     

In silico epitope-based vaccine design against influenza a neuraminidase protein: Computational analysis established on B- and T-cell epitope predictions

ObjectiveInfluenza A virus belongs to the most studied virus and its mutant initiates epidemic and pandemics outbreaks. Inoculation is the significant foundation to diminish the risk of infection. To prevent an incidence of influenza from the transmission, various practical approaches require more advancement and progress. More efforts and research must take in front to enhance vaccine efficacy.MethodsThe present research emphasizes the development and expansion of a universal vaccine for the influenza virus. Research focuses on vaccine design with high efficacy. In this study, numerous computational approaches were used, covering a wide range of elements and ideas in bioinformatics methodology. Various B and T-cell epitopic peptides derived from the Neuraminidase protein N1 are recognized by these approaches. With the implementation of numerous obtained databases and bioinformatics tools, the different immune framework methods of the conserved sequences of N1 neuraminidase were analyzed. NCBI databases were employed to retrieve amino acid sequences. The antigenic nature of the neuraminidase sequence was achieved by the VaxiJen server and Kolaskar and Tongaonkar method. After screening of various B and T cell epitopes, one efficient peptide each from B cell epitope and T cell epitopes was assessed for their antigenic determinant vaccine efficacy. Identical two B cell epitopes were recognized from the N1 protein when analyzed using B-cell epitope prediction servers. The detailed examination of amino acid sequences for interpretation of B and T cell epitopes was achieved with the help of the ABCPred and Immune Epitope Database.ResultsComputational immunology via immunoinformatic study exhibited RPNDKTG as having its high conservancy efficiency and demonstrated as a good antigenic, accessible surface hydrophilic B-cell epitope. Among T cell epitope analysis, YVNISNTNF was selected for being a conserved epitope. T cell epitope was also analyzed for its allergenicity and cytotoxicity evaluation. YVNISNTNF epitope was found to be a non-allergen and not toxic for cells as well. This T-cell epitope with maximum world populace coverages was scrutinized for its association with the HLA-DRB1*0401 molecule. Results from docking simulation analyses showed YVNISNTNF having lower binding energy, the radius of gyration (Rg), RMSD values, and RMSE values which make the protein structure more stable and increase its ability to become an epitopic peptide for influenza virus vaccination.ConclusionsWe propose that this epitope analysis may be successfully used as a measurement tool for the robustness of an antigen–antibody reaction between mutant strains in the annual design of the influenza vaccine.     

Identification of a linear epitope of interferon-alpha2b recognized by neutralizing monoclonal antibodies.

Four monoclonal antibodies (mAbs) directed against the recombinant human interferon-alpha2b (IFN-alpha2b) were used as probes to study the interaction of the IFN molecule to its receptors. The [125I]IFN-alpha2b binding to immobilized mAbs was completely inhibited by IFN-alpha2b and IFN-alpha2a but neither IFNbeta nor IFNgamma showed any effect. Gel-filtration HPLC of the immune complexes formed by incubating [125I]IFN-alpha2b with paired mAbs revealed the lack of simultaneous binding of two different antibodies to the tracer, suggesting that all mAbs recognize the same IFN antigenic domain. Furthermore, the mAbs were also able to neutralize the IFN-alpha2b anti-viral and anti-proliferative activities as well as [125I]IFN-alpha2b binding to WISH cell-membranes. As [125I]mAbs did not recognize IFN exposed epitopes in the IFN:receptor complexes, mAb induction of a conformational change in the IFN binding domain impairing its binding to receptors was considered unlikely. In order to identify the IFN region recognized by mAbs, IFN-alpha2b was digested with different proteolytic enzymes. Immunoreactivity of the resulting peptides was examined by Western blot and their sequences were established by Edman degradation after blotting to poly(vinylidene difluoride) membranes. Data obtained indicated that the smallest immunoreactive region recognized by mAbs consisted of residues 107-132 or 107-146. As this zone includes the sequence 123-140, which has been involved in the binding to receptors, and our mAbs did not show an allosteric behaviour, it is concluded that they are directed to overlapping epitopes located close to or even included in the IFN binding domain.     

Characterization of a high-affinity monoclonal antibody to calcineurin whose epitope defines a new structural domain of calcineurin A

Monoclonal antibodies have been raised against native calcineurin using conventional in vivo immunization and hybridoma procedures. The relatively high affinity of nonimmune IgG for the two subunits of calcineurin resulted in large nonspecific binding values for immunoassays of native, dissociated and denatured calcineurin, which complicated the antibody screening. Monoclonal aCn5, a high-affinity IgG1 that exhibits specific binding, was characterized. Other calmodulin-binding proteins tested were not recognized by aCn5. Simple binding properties were exhibited in solid-phase experiments, Kd = 26 (+/- 4) pM, but the stoichiometry was low. The loss of immunoreactivity after denaturation of calcineurin indicated that the aCn5 epitope is of the assembled topographic, not segmental, type. The epitope was located to the A subunit and affinity was unaffected by the presence of calcineurin B. The epitope remained intact after proteolytic removal of the amino-terminal 20 residues of calcineurin A essential for phosphatase activity, and the carboxyl-terminal inhibitory and calmodulin-binding domains. The calmodulin-binding peptide derived from calcineurin, cA8, was not recognized by aCn5. Addition of Ca2+, Mn2+, Ni2+, chelators or dithiothreitol did not influence the affinity of aCn5 for the holoenzyme. Phosphatase activity of calcineurin, in the presence and absence of calmodulin and after removal of the inhibitory domain, was little affected by aCn5. Thus, the aCn5 epitope defines a previously unidentified structural domain of calcineurin A located in a region of the proteolytically resistant core that is topologically distinct from the catalytic, inhibitory, calmodulin-binding and calcineurin-B-binding domains, and not functionally connected with calcineurin B or the putative metal-binding domain(s).     

Computational design of a water-soluble analog of phospholamban

Membrane proteins and water-soluble proteins share a similar core. This similarity suggests that it should be possible to water-solubilize membrane proteins by mutating only their lipid-exposed residues. We have developed computational tools to design water-soluble variants of helical membrane proteins, using the pentameric phospholamban (PLB) as our test case. To water-solublize PLB, the membrane-exposed positions were changed to polar or charged amino acids, while the putative core was left unaltered. We generated water-soluble phospholamban (WSPLB), and compared its properties to its predecessor PLB. In aqueous solution, WSPLB mimics all of the reported properties of PLB including oligomerization state, helical structure, and stabilization upon phosphorylation. We also characterized the truncated mutant WSPLB (21-52) comprising only the former transmembrane segment of PLB. This peptide shows a decreased specificity for forming a pentameric oligomerization state.     

RosettaRemodel: a generalized framework for flexible backbone protein design

We describe RosettaRemodel, a generalized framework for flexible protein design that provides a versatile and convenient interface to the Rosetta modeling suite. RosettaRemodel employs a unified interface, called a blueprint, which allows detailed control over many aspects of flexible backbone protein design calculations. RosettaRemodel allows the construction and elaboration of customized protocols for a wide range of design problems ranging from loop insertion and deletion, disulfide engineering, domain assembly, loop remodeling, motif grafting, symmetrical units, to de novo structure modeling.     

BON-BONs: cyclic molecules with a boron-oxygen-nitrogen backbone. Computational studies of their thermodynamic properties

Although they were first reported in 1963, molecules with a boron-oxygen-nitrogen dimeric backbone do not seem to have been investigated seriously in terms of thermodynamic properties. Here we report on the calculated structures and properties, including thermodynamics, of several so-called “BON-BON” molecules. With the popularity of nitrogen-containing substituents on new high-energy materials, nitro-substituted BON-BONs were a focus of our investigation. A total of 42 BON-BON molecules were evaluated, and thermochemical analysis shows a decrease in the specific enthalpy of combustion or decomposition with increasing NO₂ content, consistent with other systems.     

A high-affinity natural autoantibody from human cord blood defines a physiologically relevant epitope on the FcepsilonRIalpha

Natural Abs represent the indigenous immune repertoire and are thus present at birth and persist throughout life. Previously, human autoantibodies to the alpha domain of the high-affinity IgE receptor (FcepsilonRIalpha) have been isolated from Ab libraries derived from normal donors and patients with chronic urticaria. To investigate whether these anti-FcepsilonRIalpha Abs are present in the germline repertoire, we constructed a phage Fab display library from human cord blood, which represents the naive immune repertoire before exposure to exogenous Ags. All isolated clones specific to the FcepsilonRIalpha had the same sequence. This single IgM Ab, named CBMalpha8, was strictly in germline configuration and had high affinity and functional in vitro anaphylactogenic activity. Inhibition experiments indicated an overlapping epitope on the FcepsilonRIalpha recognized by both CBMalpha8 and the previously isolated anti-FcepsilonRIalpha Abs from autoimmune and healthy donors. This common epitope on FcepsilonRIalpha coincides with the binding site for IgE. Affinity measurements demonstrated the presence of Abs showing CBMalpha8-like specificity, but with a significantly lower affinity in i.v. Ig, a therapeutic multidonor IgG preparation. We propose a hypothesis of escape mutants, whereby the resulting lower affinity IgG anti-FcepsilonRIalpha Abs are rendered less likely to compete with IgE for binding to FcepsilonRIalpha.     

Computational design of a substrate specificity mutant of a protein

The wild-type trp repressor of E. coli bound 5-methoxytryptophan, a Trp analogue, less tightly than Trp. A mutant repressor (Val⁵⁸→Ala) that should bind 5-methoxytryptophan preferentially to Trp was computationally designed by free-energy calculations accompanied by free-energy decomposition. The designed mutant was demonstrated by experiments to bind 5-methoxytryptophan more tightly than Trp, consistent with the computational prediction. This success indicates the usefulness of free energy decomposition in protein design. Proteins 26:459–464 © 1996 Wiley-Liss, Inc.     

Fine mapping and interaction analysis of a linear rabies virus neutralizing epitope

A novel human antibody AR16, targeting the G5 linear epitope of rabies virus glycoprotein (RVG) was shown to have promising antivirus potency. Using AR16, the minimal binding region within G5 was identified as HDFR (residues 261–264), with key residues HDF (residues 261–263) identified by alanine replacement scanning. The key HDF was highly conserved within phylogroup I Lyssaviruses but not those in phylogroup II. Using computer-aided docking and interaction models, not only the key residues (Asp30, Asp31, Tyr32, Trp53, Asn54, Glu99, Ile101, and Trp166) of AR16 that participated in the interaction with G5 were identified, the van der Waals forces that mediated the epitope–antibody interaction were also revealed. Seven out of eight presumed key residues (Asp30, Asp31, Tyr32, Trp53, Asn54, Glu99, and Ile101) were located at the variable regions of AR16 heavy chains. A novel mAb cocktail containing AR16 and CR57, has the potential to recognize non-overlapping, non-competing epitopes, and neutralize a broad range of rabies virus.