Studies of Human Interleukin 24

In this report we present our progress in the functional and structural studies of human interleukin 24 (hIL-24). Interleukins and interferons (cytokines) together with their cognate receptors form a `front-end' of convoluted signaling networks, responsible for an immune-response to the presence of various pathogens. Cytokines are subjected to vigorous research related to their role in human physiology and disease and potential therapeutic uses. After many years of intense studies, information on some of cytokines is absent or limited, partly because new members are still being identified as well as due to difficulties of generating significant amounts of active preparations. A sub-family of `IL-10-related cytokines' also called the cytokine receptor type 2 family (CRF2) comprises nine members. One of CRF2 members is interleukin 24 (IL-24). The most interesting biological feature of human IL-24 (hIL-24) is its tumor inhibitory activity, observed in vitro for several cancer cell lines. IL-24 signals through the receptor complex comprising the high-affinity chain (IL-22R1 or IL-20R1) and the secondary, low-affinity chain (IL-20R2). Structure of IL-24 is currently unknown, although it is expected to be similar to those of other members of the CRF2 family. However, in silica analysis indicates several differences in the molecule of IL-24 when compared to its close homologues. One of more interesting is lack of two canonical disulfide bonds, found in all other interleukins from the CRF2 family, due to missing needed Cys residues. While an alternative, unique disulfide bond in IL-24 is possible, the appropriate experimental evidence is missing. Native IL-24 is N-glycosylated. Although this modification appears dispensable for biological activity of related cytokines, its role in IL-24 is not clear. We have expressed insoluble hIL-24 in bacteria and refolded it subsequently into a soluble, functional form. The mass spectrometry analysis confirmed presence of a single disulfide bond. Our non-glycosylated variant of hIL-24 activates the cognate receptor as efficiently as commercial preparations from eukaryotic sources. We observed, however, that stability of refolded hIL-24 is somewhat compromised. Subsequently, we plan investigating the structural properties of this cytokine.

25 years of crystallographic studies related to HIV/AIDS

This year we celebrate 25 years since the first crystal structures of HIV-encoded proteins became available. The structure of HIV protease, the first one to be determined, immediately became a guide for designing drugs directed at this enzyme crucial to viral maturation, with several drugs gaining approval in a record time of 6 years. The structures of the other HIV enzymes (reverse transcriptase, RNase H, and integrase) followed and all were immediately useful to drug developers. Other HIV-encoded proteins that control membrane fusion, viral entry, and regulatory processes were also pursued, together with host proteins that are involved in maintaining viral life-cycle. This large body of structural knowledge was crucial to the development of multi-drug therapy that changed the face of the AIDS epidemic from an irrevocably mortal disease to a manageable infection. The history of these 25 years of world-wide crystallographic efforts is worth recounting.

Complexes of Fabs with a mimetic of the HIV-1 gp41 trimeric coiled-coil.

A series of mini-antibodies (monovalent and bivalent Fabs) targeting the conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 has been previously constructed and reported. Crystal structures of two closely related monovalent Fabs, one (Fab 8066) broadly neutralizing across a wide panel of HIV-1 subtype B and C viruses, and the other (Fab 8062) non-neutralizing, representing the extremes of this series, were previously solved as complexes with 5-Helix, a gp41 pre-hairpin intermediate mimetic. Binding of these Fabs to covalently stabilized chimeric trimers of N-peptides of HIV-1 gp41 (named (CCIZN36)3 or 3-H) has now been investigated using X-ray crystallography, cryo-electron microscopy, and a variety of biophysical methods. Crystal structures of the complexes between 3-H and Fab 8066 and Fab 8062 were determined at 2.8 and 3.0 Å resolution, respectively. Although the structures of the complexes with the neutralizing Fab 8066 and its non-neutralizing counterpart Fab 8062 were generally similar, small differences between them could be correlated with the biological properties of these antibodies. The conformations of the corresponding CDRs of each antibody in the complexes with 3-H and 5-Helix are very similar. The adaptation to a different target upon complex formation is predominantly achieved by changes in the structure of the trimer of N-HR helices, as well as by adjustment of the orientation of the Fab molecule relative to the N-HR in the complex, via rigid-body movement. The structural data presented here indicate that binding of three Fabs 8062 with high affinity requires more significant changes in the structure of the N-HR trimer compared to binding of Fab 8066. A comparative analysis of the structures of Fabs complexed to different gp41 intermediate mimetics allows further evaluation of biological relevance for generation of neutralizing antibodies, as well as provides novel structural insights into immunogen design.