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The sera genes of the malaria-causing parasite Plasmodium encode a family of unique proteins that are maximally expressed at the time of egress of parasites from infected red blood cells. These multi-domain proteins are unique, containing a central papain-like cysteine-protease fragment enclosed between the disulfide-linked N- and C-terminal domains. However, the central fragment of several members of this family, including serine repeat antigen 5 (SERA5), contains a serine (S596) in place of the active-site cysteine. Here we report the crystal structure of the central protease-like domain of Plasmodium falciparum SERA5, revealing a number of anomalies in addition to the putative nucleophilic serine: (1) the structure of the putative active site is not conducive to binding substrate in the canonical cysteine-protease manner; (2) the side chain of D594 restricts access of substrate to the putative active site; and (3) the S(2) specificity pocket is occupied by the side chain of Y735, reducing this site to a small depression on the protein surface. Attempts to determine the structure in complex with known inhibitors were not successful. Thus, despite having revealed its structure, the function of the catalytic domain of SERA5 remains an enigma.

The serine repeat antigen (SERA) proteins of the malaria parasitesPlasmodiumspp. contain a putative enzyme domain similar to that of papain family cysteine proteases. InPlasmodium falciparumparasites, more than half of the SERA family proteins, including the most abundantly expressed form, SERA5, have a cysteine-to-serine substitution within the putative catalytic triad of the active site. Although SERA5 is required for blood-stage parasite survival, the occurrence of a noncanonical catalytic triad casts doubt on the importance of the enzyme domain in this function. We used phage display to identify a small (14-residue) disulfide-bonded cyclic peptide (SBP1) that targets the enzyme domain of SERA5. Biochemical characterization of the interaction shows that it is dependent on the conformation of both the peptide and protein. Addition of this peptide to parasite cultures compromised development of late-stage parasites compared to that of control parasites or those incubated with equivalent amounts of the carboxymethylated peptide. This effect was similar in two different strains ofP. falciparumas well as in a transgenic strain where the gene encoding the related serine-type parasitophorous vacuole protein SERA4 was deleted. In compromised parasites, the SBP1 peptide crosses both the erythrocyte and parasitophorous vacuole membranes and accumulates within the parasitophorous vacuole. In addition, both SBP1 and SERA5 were identified in the parasite cytosol, indicating that the plasma membrane of the parasite was compromised as a result of SBP1 treatment. These data implicate an important role for SERA5 in the regulation of the intraerythrocytic development of late-stage parasites and as a target for drug development.

The structure of the complex between a recombinant single-chain Fv construct of antibody NC10 with a five-residue peptide linker between VH and VL (termed scFv(5)), and its antigen, tetrameric neuraminidase from influenza virus (NA), has been determined and refined at 2.5 A resolution. The antibody-antigen binding interface is very similar to that of a similar NC10 scFv-NA complex in which the scFv has a 15-residue peptide linker (scFv(15)), and the NC10 Fab-NA complex. However, scFv(5) and scFv(15) have different stoichiometries in solution. While scFv(15) is predominantly monomeric in solution, scFv(5) forms dimers exclusively, because the five-residue linker is not long enough to permit VH and VL domains from the same polypeptide associating and forming an antigen-binding site. Upon forming a complex with NA, scFv(15) forms a approximately 300 kDa complex corresponding to one NA tetramer binding four scFv(15) monomers, while scFv(5) forms a approximately 590 kDa complex, corresponding to two NA tetramers crosslinked by four bivalent scFv(5) dimers. However, the dimeric scFv(5) in the scFv(5)-NA crystals does not crosslink NA tetramers, and modelling studies indicate that it is not possible to pack four dimeric and simultaneously bivalent scFvs between the NA tetramers with only a five-residue linker between VH and VL. The inability arises from the exacting requirement to orient the two antigen-binding surfaces to bind the tetrameric NA antigen while avoiding steric clashes with NC10 scFv(5) dimers bound to other sites on the NA tetramer. The utility of bivalent or bifunctional scFvs with short linkers may therefore be restricted by the steric constraints imposed by binding multivalent antigens.

Background While it is well known that different antibodies can be produced against a particular antigen, and even against a particular site on an antigen, up until now there have been no structural studies of cross-reacting antibodies of this type. One antibody– antigen complex whose structure is known is that of the influenza virus antigen, neuraminidase, in complex with the NC41 antibody. Another anti-neuraminidase antibody, NC10, binds to an overlapping site on the antigen. The structure of the complex formed by this antibody with neuraminidase is described here and compared with the NC41-containing complex. Results The crystal structure of the NC10 Fab– neuraminidase complex has been refined to a nominal resolution of 2.5a. Approximately 80% of the binding site of the NC10 antibody on neuraminidase overlaps with that of the NC41 antibody. The epitope residues of neuraminidase are often engaged in quite different interactions with the two antibodies. Although the NC10 and NC41 antibodies have identical amino acid sequences within the first complementarity determining region of their heavy chains, this is not the basis of the cross-reaction. Conclusions The capacity of two different proteins to bind to the same target structure on a third protein need not be based on the existence of identical or homologous amino acid sequences within those proteins. As we have demonstrated, amino acid residues on the common target structure may be in quite different chemical environments, and may also adopt different conformations within two protein– protein complexes.

Background N -acetylneuraminate lyase catalyzes the cleavage of N -acetylneuraminic acid (sialic acid) to form pyruvate and N - acetyl- d -mannosamine. The enzyme plays an important role in the regulation of sialic acid metabolism in bacteria. The reverse reaction can be exploited for the synthesis of sialic acid and some of its derivatives. Results The structure of the enzyme from Escherichia coli has been determined to 2.2 a resolution by X-ray crystallography. The enzyme is shown to be a tetramer, in which each subunit consists of an α / β -barrel domain followed by a carboxy-terminal extension of three α -helices. Conclusions The active site of the enzyme is tentatively identified as a pocket at the carboxy-terminal end of the eight- stranded β -barrel. Lys165 lies within this pocket and is probably the reactive residue which forms a Schiff base intermediate with the substrate. The sequence of N - acetylneuraminate lyase has similarities to those of dihydrodipicolinate synthase and MosA (an enzyme implicated in rhizopine synthesis) suggesting that these last two enzymes share a similar structure to N -acetylneuraminate lyase.

The single-chain antibody variable fragment (scFv), with a 15-residue polypeptide linker (Gly4Ser)3, of monoclonal antibody NC10 was expressed in Escherichia coli and purified to homogeneity. This scFv molecule, refolded from 6 M guanidine hydrochloride, was predominantly a monomer of 27 kDa and was stable on storage at 4 degrees and 20 degrees C. At higher protein concentrations (approximately 5 mg/ml) dimer and higher-molecular-mass multimers were formed and freezing enhanced this aggregation. The dimer was not stable and dissociated to monomer at 20 degrees C with a half-life of approximately 8 days. The higher-molecular-mass multimers and dimer dissociated to monomer in 60% ethylene glycol. Both the monomer and dimer were active and with tern N9 sialidase yielded complexes of 276 kDa and 569 kDa, respectively, indicating that four scFv molecules bound/sialidase tetramer and that the dimer was bivalent and cross-linked two sialidase tetramers. Binding studies at low concentrations and using radiolabelled scFv indicated that the binding affinity of the dimer was approximately twofold higher than that of the monomer, and the binding affinities of the scFv were similar to that of the parent NC10 antigen-binding fragment (Fab) molecule. A complex between tern N9 sialidase and NC10 scFv was crystallized and the structure of the complex was solved at 0.3-nm resolution by X-ray diffraction. Comparison of this scFv/sialidase structure with the parent Fab/sialidase structure revealed that the modes of attachment of scFv and Fab to sialidase were very similar. There was no discernible electron density for the peptide linker joining the variable heavy (VH) and variable light (VL) chains. A close interaction between two symmetry-related scFv suggests that they may have crystallized as dimers.

Phagemid vectors have been developed which promise to supersede hybridoma technology for the selection and production of human antibodies. We have modified an existing phagemid vector to improve the stability of synthesized soluble antibody fragments. The vector allows the antibody fragment to be produced: i) as a soluble protein incorporating a stable carboxyl terminal octapeptide (FLAG) or, ii) on the surface of a bacteriophage fused to a minor coat protein (the gene III protein). The antibody gene encoding the well characterized monoclonal antibody NC10 (an antibody that recognizes the neuraminidase of the influenza strain N9) was inserted as a single chain Fv construct into the phagemid vectors pHFA and pHFA/SacII. Western blotting, ELISA and electron microscopy studies showed that recombinant clones could be manipulated to either synthesize soluble protein into the periplasm or present the protein on the surface of bacteriophage. Cosynthesis of GroEL and GroES chaperonins resulted in complete proteolysis of the scFvNC10-FLAG-gIIIp fusion product and did not improve total phage production. Coexpression of chaperonins should be used with caution for library construction due to the expected selection pressure for protease resistant gene III fusions.

Phosphomannomutase (PMM) catalyses the conversion of mannose-6-phosphate to mannose-1-phosphate, an essential step in mannose activation and the biosynthesis of glycoconjugates in all eukaryotes. Deletion of PMM from Leishmania mexicana results in loss of virulence, suggesting that PMM is a promising drug target for the development of anti-leishmanial inhibitors. We report the crystallization and structure determination to 2.1 A of L. mexicana PMM alone and in complex with glucose-1,6-bisphosphate to 2.9 A. PMM is a member of the haloacid dehalogenase (HAD) family, but has a novel dimeric structure and a distinct cap domain of unique topology. Although the structure is novel within the HAD family, the leishmanial enzyme shows a high degree of similarity with its human isoforms. We have generated L. major PMM knockouts, which are avirulent. We expressed the human pmm2 gene in the Leishmania PMM knockout, but despite the similarity between Leishmania and human PMM, expression of the human gene did not restore virulence. Similarities in the structure of the parasite enzyme and its human isoforms suggest that the development of parasite-selective inhibitors will not be an easy task.

The variable heavy (VH) and variable light (VL) genes of NC10, a monoclonal antibody with specificity toward N9 neuraminidase (NA), were cloned and sequenced. A single chain Fv (scFv) fragment of NC10, consisting of VH and VL domains joined by a peptide linker, was designed, constructed and expressed in the E. coli expression vector pPOW. The N-terminal secretion signal PelB directed the synthesized protein into the periplasm where it was associated with the insoluble membrane fraction. An octapeptide (FLAG) tail was fused to the C-terminus of the single chain Fv to aid in its detection and remained intact throughout the protein purification process. NC10 scFv was purified by solubilization of the E. coli membrane fraction with guanidinium hydrochloride followed by column chromatography. The purified NC10 scFv showed binding affinity for its antigen, NA, 2-fold lower than that of the parent Fab. The complex between NA and the scFv has been crystallized by the vapor diffusion method. The crystals are tetragonal, space group P42(1)2, with unit cell dimensions a = b = 141 A, c = 218 A.