Bankala Krishnarjuna

Conformational Dynamics and Antigenicity in the Disordered Malaria Antigen Merozoite Surface Protein 2

Merozoite surface protein 2 (MSP2) of Plasmodium falciparum is an abundant, intrinsically disordered protein that is GPI-anchored to the surface of the invasive blood stage of the malaria parasite. Recombinant MSP2 has been trialled as a component of a malaria vaccine, and is one of several disordered proteins that are candidates for inclusion in vaccines for malaria and other diseases. Nonetheless, little is known about the implications of protein disorder for the development of an effective antibody response. We have therefore undertaken a detailed analysis of the conformational dynamics of the two allelic forms of MSP2 (3D7 and FC27) using NMR spectroscopy. Chemical shifts and NMR relaxation data indicate that conformational and dynamic properties of the N- and C-terminal conserved regions in the two forms of MSP2 are essentially identical, but significant variation exists between and within the central variable regions. We observe a strong relationship between the conformational dynamics and the antigenicity of MSP2, as assessed with antisera to recombinant MSP2. Regions of increased conformational order in MSP2, including those in the conserved regions, are more strongly antigenic, while the most flexible regions are minimally antigenic. This suggests that modifications that increase conformational order may offer a means to tune the antigenicity of MSP2 and other disordered antigens, with implications for vaccine design.

Structural basis for epitope masking and strain specificity of a conserved epitope in an intrinsically disordered malaria vaccine candidate

Merozoite surface protein 2 (MSP2) is an intrinsically disordered, membrane-anchored antigen of the malaria parasite Plasmodium falciparum. MSP2 can elicit a protective, albeit strain-specific, antibody response in humans. Antibodies are generated to the conserved N- and C-terminal regions but many of these react poorly with the native antigen on the parasite surface. Here we demonstrate that recognition of a conserved N-terminal epitope by mAb 6D8 is incompatible with the membrane-bound conformation of that region, suggesting a mechanism by which native MSP2 escapes antibody recognition. Furthermore, crystal structures and NMR spectroscopy identify transient, strain-specific interactions between the 6D8 antibody and regions of MSP2 beyond the conserved epitope. These interactions account for the differential affinity of 6D8 for the two allelic families of MSP2, even though 6D8 binds to a fully conserved epitope. These results highlight unappreciated mechanisms that may modulate the specificity and efficacy of immune responses towards disordered antigens.

Structure and dynamics of apical membrane antigen 1 from Plasmodium falciparum FVO

Apical membrane antigen 1 (AMA1) interacts with RON2 to form a protein complex that plays a key role in the invasion of host cells by malaria parasites. Blocking this protein-protein interaction represents a potential route to controlling malaria and related parasitic diseases, but the polymorphic nature of AMA1 has proven to be a major challenge to vaccine-induced antibodies and peptide inhibitors exerting strain-transcending inhibitory effects. Here we present the X-ray crystal structure of AMA1 domains I and II from Plasmodium falciparum strain FVO. We compare our new structure to those of AMA1 from P. falciparum 3D7 and Plasmodium vivax. A combination of normalized B factor analysis and computational methods has been used to investigate the flexibility of the domain I loops and how this correlates with their roles in determining the strain specificity of human antibody responses and inhibitory peptides. We also investigated the domain II loop, a key region involved in inhibitor binding, by comparison of multiple AMA1 crystal structures. Collectively, these results provide valuable insights that should contribute to the design of strain-transcending agents targeting P. falciparum AMA1.

Strain-transcending immune response generated by chimeras of the malaria vaccine candidate merozoite surface protein 2.

MSP2 is an intrinsically disordered protein that is abundant on the merozoite surface and essential to the parasite Plasmodium falciparum. Naturally-acquired antibody responses to MSP2 are biased towards dimorphic sequences within the central variable region of MSP2 and have been linked to naturally-acquired protection from malaria. In a phase IIb study, an MSP2-containing vaccine induced an immune response that reduced parasitemias in a strain-specific manner. A subsequent phase I study of a vaccine that contained both dimorphic forms of MSP2 induced antibodies that exhibited functional activity in vitro. We have assessed the contribution of the conserved and variable regions of MSP2 to the generation of a strain-transcending antibody response by generating MSP2 chimeras that included conserved and variable regions of the 3D7 and FC27 alleles. Robust anti-MSP2 antibody responses targeting both conserved and variable regions were generated in mice, although the fine specificity and the balance of responses to these regions differed amongst the constructs tested. We observed significant differences in antibody subclass distribution in the responses to these chimeras. Our results suggest that chimeric MSP2 antigens can elicit a broad immune response suitable for protection against different strains of P. falciparum.

Solution NMR characterization of apical membrane antigen 1 and small molecule interactions as a basis for designing new antimalarials

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) plays an important role in the invasion by merozoites of human red blood cells during a malaria infection. A key region of PfAMA1 is a conserved hydrophobic cleft formed by 12 hydrophobic residues. As anti‐apical membrane antigen 1 antibodies and other inhibitory molecules that target this hydrophobic cleft are able to block the invasion process, PfAMA1 is an attractive target for the development of strain‐transcending antimalarial agents. As solution nuclear magnetic resonance spectroscopy is a valuable technique for the rapid characterization of protein–ligand interactions, we have determined the sequence‐specific backbone assignments for PfAMA1 from two P. falciparum strains, FVO and 3D7. Both selective labelling and unlabelling strategies were used to complement triple‐resonance experiments in order to facilitate the assignment process. We have then used these assignments for mapping the binding sites for small molecules, including benzimidazoles, pyrazoles and 2‐aminothiazoles, which were selected on the basis of their affinities measured from surface plasmon resonance binding experiments. Among the compounds tested, benzimidazoles showed binding to a similar region on both FVO and 3D7 PfAMA1, suggesting that these compounds are promising scaffolds for the development of novel PfAMA1 inhibitors. Copyright

Structure, folding and stability of a minimal homologue from Anemonia sulcata of the sea anemone potassium channel blocker ShK

Graphical abstract AsK132958 is a 29‐residue peptide identified in a transcriptomic study of Anemonia sulcata. It has the same disulfide framework and a similar structure to ShK. AsK132958 is not active against KV1.3 channels, owing to the lack of a Lys‐Tyr dyad and other functionally important amino acid residues. AsK132958 is more resistant to proteolysis than ShK. Introducing a Lys‐Tyr functional dyad to the AsK132958 structural scaffold may be a useful way of developing a proteolytically stable KV1.3 blocker. Figure. No caption available. HighlightsAsK132958 is one of the shortest peptides with a ShK/BgK‐like cysteine framework.AsK132958 is a structural homologue of ShK.Despite having an ShK‐like scaffold, AsK132958 is not active against KV1.3 or related potassium channels.AsK132958 is more resistant to proteases than ShK and is a promising scaffold for engineering other activities.AsK132958 could be an evolutionary precursor of peptides with ShK‐like scaffold and activity. ABSTRACT Peptide toxins elaborated by sea anemones target various ion‐channel sub‐types. Recent transcriptomic studies of sea anemones have identified several novel candidate peptides, some of which have cysteine frameworks identical to those of previously reported sequences. One such peptide is AsK132958, which was identified in a transcriptomic study of Anemonia sulcata and has a cysteine framework similar to that of ShK from Stichodactyla helianthus, but is six amino acid residues shorter. We have determined the solution structure of this novel peptide using NMR spectroscopy. The disulfide connectivities and structural scaffold of AsK132958 are very similar to those of ShK but the structure is more constrained. Toxicity assays were performed using grass shrimp (Palaemonetes sp) and Artemia nauplii, and patch‐clamp electrophysiology assays were performed to assess the activity of AsK132958 against a range of voltage‐gated potassium (KV) channels. AsK132958 showed no activity against grass shrimp, Artemia nauplii, or any of the KV channels tested, owing partly to the absence of a functional Lys‐Tyr dyad. Three AsK132958 analogues, each containing a Tyr in the vicinity of Lys19, were therefore generated in an effort to restore binding, but none showed activity against any of KV channels tested. However, AsK132958 and its analogues are less susceptible to proteolysis than that of ShK. Our structure suggests that Lys19, which might be expected to occupy the pore of the channel, is not sufficiently accessible for binding, and therefore that AsK132958 must have a distinct functional role that does not involve KV channels.

Structure and Characterisation of a Key Epitope in the Conserved C-Terminal Domain of the Malaria Vaccine Candidate MSP2

Merozoite surface protein 2 (MSP2) is an intrinsically disordered antigen that is abundant on the surface of the malaria parasite Plasmodium falciparum. The two allelic families of MSP2, 3D7 and FC27, differ in their central variable regions, which are flanked by highly conserved C-terminal and N-terminal regions. In a vaccine trial, full-length 3D7 MSP2 induced a strain-specific protective immune response despite the detectable presence of conserved region antibodies. This work focuses on the conserved C-terminal region of MSP2, which includes the only disulphide bond in the protein and encompasses key epitopes recognised by the mouse monoclonal antibodies 4D11 and 9H4. Although the 4D11 and 9H4 epitopes are overlapping, immunofluorescence assays have shown that the mouse monoclonal antibody 4D11 binds to MSP2 on the merozoite surface with a much stronger signal than 9H4. Understanding the structural basis for this antigenic difference between these antibodies will help direct the design of a broad-spectrum and MSP2-based malaria vaccine. 4D11 and 9H4 were reengineered into antibody fragments [variable region fragment (Fv) and single-chain Fv (scFv)] and were validated as suitable models for their full-sized IgG counterparts by surface plasmon resonance and isothermal titration calorimetry. An alanine scan of the 13-residue epitope 3D7-MSP2207-222 identified the minimal binding epitope of 4D11 and the key residues involved in binding. A 2.2-Å crystal structure of 4D11 Fv bound to the eight-residue epitope NKENCGAA provided valuable insight into the possible conformation of the C-terminal region of MSP2 on the parasite. This work underpins continued efforts to optimise recombinant MSP2 constructs for evaluation as potential vaccine candidates.

NMR Structure Implications of Enhanced Efficacy of Obestatin Fragment Analogs

Obestatin is a more recently discovered hormone that is encoded by the ghrelin gene and produced in the stomach and gut. We report NMR analysis on synthetic Obestatin (OB23), a 23 residue peptide, along with three overlapping fragments of the same in methanol solvent as a first step towards structure activity relationship. Selective substitutions on the promising N-terminal and middle fragments of obestatin have been carried out in order to improve the efficacy and potency. In the N-terminal fragment two peptides were obtained by the replacement of Gly (8) with α-aminoisobutyric acid (Aib, U) and Phe (F5) with Cyclohexylalanine (Cha). In case of the middle fragment both Gly (3) and Gly (8) were replaced with Aib residues. The rationale being, these unusual amino acids could provide protection from immediate degradation and aid structure stabilization. Our previous studies showed that the N-terminal and the middle fragment were unstructured and hence this substitution would directly evaluate the effect of structure on the activity of these fragment analogs. Detailed NMR analysis clearly demonstrates formation of helical secondary structure in all the peptide analogues and provides justification for relative activities reported by our group previously (Nagaraj et al. 2009).

Lipid interactions modulate the structural and antigenic properties of the C‐terminal domain of the malaria antigen merozoite surface protein 2

Merozoite surface protein 2 (MSP2) is a highly abundant, GPI‐anchored antigen on the malaria parasite Plasmodium falciparum. MSP2 induces an immune response in the context of natural infections and vaccine trials, and these responses are associated with protection from parasite infection. Recombinant MSP2 is highly disordered in solution but antigenic analyses suggest that it is more ordered on the merozoite surface. We have shown previously that the interaction of recombinant full‐length MSP2 with lipid surfaces induces a conformational change in the conserved N‐terminal region of MSP2, which contributes to epitope masking in this region. To explore the impacts of lipid interactions on the conformation and antigenicity of the conserved C‐terminal region of MSP2, a construct corresponding to this domain, MSP2172–221, was designed. NMR studies indicate that many residues in MSP2172–221 interact with DPC micelles, including some in epitopes recognised by C‐terminal‐specific monoclonal antibodies, but, in contrast to the MSP2 N‐terminus, there is no indication of stable helical conformation. The binding affinities of a panel of monoclonal antibodies indicate that MSP2172–221 is antigenically similar to full‐length MSP2 and show that liposome conjugation alters the antigenicity in a manner that may mimic native MSP2 on the merozoite surface. These findings highlight the impact of lipid interactions on the conformation and antigenicity of MSP2172–221 and will assist in the design of recombinant MSP2 immunogens for use as malaria vaccine candidates.

Does Aluminium Bind to Histidine? An NMR Investigation of Amyloid β12 and Amyloid β16 Fragments

Aluminium and zinc are known to be the major triggering agents for aggregation of amyloid peptides leading to plaque formation in Alzheimers disease. While zinc binding to histidine in Aβ (amyloid β) fragments has been implicated as responsible for aggregation, not much information is available on the interaction of aluminium with histidine. In the NMR study of the N‐terminal Aβ fragments, DAEFRHDSGYEV (Aβ12) and DAEFRHDSGYEVHHQK (Aβ16) presented here, the interactions of the fragments with aluminium have been investigated. Significant chemical shifts were observed for few residues near the C‐terminus when aluminium chloride was titrated with Aβ12 and Aβ16 peptides. Surprisingly, it is nonhistidine residues which seem to be involved in aluminium binding. Based on NMR constrained structure obtained by molecular modelling, aluminium‐binding pockets in Aβ12 were around charged residues such as Asp, Glu. The results are discussed in terms of native structure propagation, and the relevance of histidine residues in the sequences for metal‐binding interactions. We expect that the study of such short amyloid peptide fragments will not only provide clues for plaque formation in aggregated conditions but also facilitate design of potential drugs for these targets.