Helical peptide arrays for lead identification and interaction site mapping.

Libraries composed of linear and cyclic peptides cannot fully represent the higher order structures of most antigenic sites. To map the binding site of ligands or antibodies, a larger part of the three-dimensional space should be sampled. Because parallel synthesis of large arrays of peptides on hydrogels is restricted to relatively small peptides, a simple and robust homodimeric helical system was chosen for antigen presentation. First, it was established in an heterodimeric system that the 26-mer peptide could be synthesized and that the helical coiled-coil peptides interact in the hydrogel in a predictable manner. Next, libraries of homodimeric coiled coils were synthesized into which the epitope was grafted. Using dedicated helical dimeric and trimeric coiled-coil libraries, the epitopes of two anti-HIV-1 gp41 monoclonal antibodies known to interact with helical structures were mapped at high resolution. These mappings precisely reflect existing X-ray data, and the arrays can be applied to lead identification, epitope mapping, and systematic analysis of amino acid contribution to coiled-coil systems.

Super Potent Bispecific Llama VHH Antibodies Neutralize HIV via a Combination of gp41 and gp120 Epitopes.

Broad and potent neutralizing llama single domain antibodies (VHH) against HIV-1 targeting the CD4 binding site (CD4bs) have previously been isolated upon llama immunization. Here we describe the epitopes of three additional VHH groups selected from phage libraries. The 2E7 group binds to a new linear epitope in the first heptad repeat of gp41 that is only exposed in the fusion-intermediate conformation. The 1B5 group competes with co-receptor binding and the 1F10 group interacts with the crown of the gp120 V3 loop, occluded in native Env. We present biophysical and structural details on the 2E7 interaction with gp41. In order to further increase breadth and potency, we constructed bi-specific VHH. The combination of CD4bs VHH (J3/3E3) with 2E7 group VHH enhanced strain-specific neutralization with potencies up to 1400-fold higher than the mixture of the individual VHHs. Thus, these new bivalent VHH are potent new tools to develop therapeutic approaches or microbicide intervention.

Epitope mapping of Ebola virus dominant and subdominant glycoprotein epitopes facilitates construction of an epitope-based DNA vaccine able to focus the antibody response in mice.

We performed epitope mapping studies on the major surface glycoprotein (GP) of Ebola virus (EBOV) using Chemically Linked Peptides on Scaffolds (CLIPS), which form linear and potential conformational epitopes. This method identified monoclonal antibody epitopes and predicted additional epitopes recognized by antibodies in polyclonal sera from animals experimentally vaccinated against or infected with EBOV. Using the information obtained along with structural modeling to predict epitope accessibility, we then constructed 2 DNA vaccines encoding immunodominant and subdominant epitopes predicted to be accessible on EBOV GP. Although a construct designed to produce a membrane-bound oligopeptide was poorly immunogenic, a construct generating a secreted oligopeptide elicited strong antibody responses in mice. When this construct was administered as a boost to a DNA vaccine expressing the complete EBOV GP gene, the resultant antibody response was focused largely toward the less immunodominant epitopes in the oligopeptide. Taken together, the results of this work suggest a utility for this method for immune focusing of antibody responses elicited by vaccination.

A structure for the yeast prohibitin complex: Structure prediction and evidence from chemical crosslinking and mass spectrometry.

The mitochondrial prohibitin complex consists of two subunits (PHB1 of 32 kD and PHB2 of 34 kD), assembled into a membrane-associated supercomplex of approximately 1 MD. A chaperone-like function in holding and assembling newly synthesized mitochondrial polypeptide chains has been proposed. To further elucidate the function of this complex, structural information is necessary. In this study we use chemical crosslinking, connecting lysine side chains, which are well scattered along the sequence. Crosslinked peptides from protease digested prohibitin complexes were identified with mass spectrometry. From these results, spatial restraints for possible protein conformation were obtained. Many interaction sites between PHB1 and PHB2 were found, whereas no homodimeric interactions were observed. Secondary and tertiary structural predictions were made using several algorithms and the models best fitting the spatial restraints were selected for further evaluation. From the structure predictions and the crosslink data we derived a structural building block of one PHB1 and one PHB2 subunit, strongly intertwined along most of their length. The size of the complex implies that approximately 14 of these building blocks are present. Each unit contains a putative transmembrane helix in PHB2. Taken together with the unit building block we postulate a circular palisade-like arrangement of the building blocks projecting into the intermembrane space.

Structure-based design for high-hanging vaccine fruits.

Although vaccines have proven life saving against a myriad of infectious diseases, various pathogens have remained refractory to prophylaxis of their host by active immunization. New insights in the three dimensional (3D) structure, domain organization and dynamics of viral and bacterial surface proteins can guide the design of effective vaccines in several ways. In this review we highlight recent developments in structure-based vaccine design that are aimed at stabilization of native conformations and focusing immune response to conserved epitopes. Detailed 3D structures of pathogen surface proteins provide knowledge on how to minimize complex antigens or how to redesign the surface of an immunogen in order to induce only relevant neutralizing antibodies against a broad range of serotypes. Structure - based vaccines with reduced complexity and broad efficacy could greatly enhance the number of people that might benefit from the therapies that are developed.

The soluble NAD+-Reducing [NiFe]-hydrogenase from Ralstonia eutropha H16 consists of six subunits and can be specifically activated by NADPH.

The soluble [NiFe]-hydrogenase (SH) of the facultative lithoautotrophic proteobacterium Ralstonia eutropha H16 has up to now been described as a heterotetrameric enzyme. The purified protein consists of two functionally distinct heterodimeric moieties. The HoxHY dimer represents the hydrogenase module, and the HoxFU dimer constitutes an NADH-dehydrogenase. In the bimodular form, the SH mediates reduction of NAD(+) at the expense of H(2). We have purified a new high-molecular-weight form of the SH which contains an additional subunit. This extra subunit was identified as the product of hoxI, a member of the SH gene cluster (hoxFUYHWI). Edman degradation, in combination with protein sequencing of the SH high-molecular-weight complex, established a subunit stoichiometry of HoxFUYHI(2). Cross-linking experiments indicated that the two HoxI subunits are the closest neighbors. The stability of the hexameric SH depended on the pH and the ionic strength of the buffer. The tetrameric form of the SH can be instantaneously activated with small amounts of NADH but not with NADPH. The hexameric form, however, was also activated by adding small amounts of NADPH. This suggests that HoxI provides a binding domain for NADPH. A specific reaction site for NADPH adds to the list of similarities between the SH and mitochondrial NADH:ubiquinone oxidoreductase (Complex I).

Mass spectrometric identification of isoforms of PR proteins in xylem sap of fungus-infected tomato.

The protein content of tomato (Lycopersicon esculentum) xylem sap was found to change dramatically upon infection with the vascular wilt fungus Fusarium oxysporum. Peptide mass fingerprinting and mass spectrometric sequencing were used to identify the most abundant proteins appearing during compatible or incompatible interactions. A new member of the PR-5 family was identified that accumulated early in both types of interaction. Other pathogenesis-related proteins appeared in compatible interactions only, concomitantly with disease development. This study demonstrates the feasibility of using proteomics for the identification of known and novel proteins in xylem sap, and provides insights into plant-pathogen interactions in vascular wilt diseases.