Cumulative phylogenetic, sequence and structural analysis of Insulin superfamily proteins provide unique structure-function insights.

The insulin superfamily proteins (ISPs), in particular, insulin, IGFs and relaxin proteins are key modulators of animal physiology. They are known to have evolved from the same ancestral gene and have diverged into proteins with varied sequences and distinct functions, but maintain a similar structural architecture stabilized by highly conserved disulphide bridges. The recent surge of sequence data and the structures of these proteins prompted a need for a comprehensive analysis, which connects the evolution of these sequences (427 sequences) in the light of available functional and structural information including representative complex structures of ISPs with their cognate receptors. This study reveals (a) unusually high sequence conservation of IGFs (>90 % conservation in 184 sequences) and provides a possible structure-based rationale for such high sequence conservation; (b) provides an updated definition of the receptor-binding signature motif of the functionally diverse relaxin family members (c) provides a probable non-canonical C-peptide cleavage site in a few insulin sequences. The high conservation of IGFs appears to represent a classic case of resistance to sequence diversity exerted by physiologically important interactions with multiple partners. We also propose a probable mechanism for C-peptide cleavage in a few distinct insulin sequences and redefine the receptor-binding signature motif of the relaxin family. Lastly, we provide a basis for minimally modified insulin mutants with potential therapeutic application, inspired by concomitant changes observed in other insulin superfamily protein members supported by molecular dynamics simulation.

High-resolution crystal structure of LpqH, an immunomodulatory surface lipoprotein of Mycobacterium tuberculosis reveals a distinct fold and a conserved cleft on its surface.

Tuberculosis, caused by Mycobacterium tuberculosis, is predominantly a disease of the lungs acquired by inhaling mycobacteria from infected individuals via airborne droplets. In order to facilitate their entry into the alveolar macrophages, mycobacteria have a collection of pathogen-associated molecular patterns (PAMPs) on their surface that are known to detect certain pattern recognition receptors present on the surface of host cells. A major group of these PAMPs includes mycobacterial lipoproteins, of which, the 19 kDa surface antigen LpqH, has been reported to play a critical role in both host-pathogen interactions as well as pleiotropic immune regulation. Despite its crucial involvement in tuberculosis, the detailed structure-function relationship of this protein remains to be explored. Here, we report the high-resolution crystal structure of the non-acylated LpqH (LpqH48-159) at a resolution of 1.26 Å, which adopts a unique fold. Flow cytometry-based experiments show that the protein can bind and induce apoptosis in PMA-activated human monocytic cell line THP-1, indicative of the preservation of functionality of the protein. Furthermore, analysis of conservation of LpqH sequences from Mycobacterium species reveals a patch of conserved residues on the surface which may play a role in its binding partner recognition and hence in host-pathogen interaction.

Structural Insights into N-terminal IgV Domain of BTNL2, a T Cell Inhibitory Molecule, Suggests a Non-canonical Binding Interface for Its Putative Receptors.

T cell costimulation is mediated by the interaction of a number of receptors and ligands present on the surface of the T cell and antigen-presenting cell, respectively. Stimulatory or inhibitory signals from these receptor-ligand interactions work in tandem to preserve immune homeostasis. BTNL2 is a type-1 membrane protein that provides inhibitory signal to T cells and plays an important role in several inflammatory and autoimmune diseases. Therefore, manipulation of the molecular interaction of BTNL2 with its putative receptor could provide strategies to restore immune homeostasis in these diseases. Hence, it is imperative to study the structural characteristics of this molecule, which will provide important insights into its function as well. In this study, the membrane-distal ectodomain of murine BTNL2 was expressed in bacteria as inclusion bodies, refolded in vitro and purified for functional and structural characterization. The domain is monomeric in solution as demonstrated by size-exclusion chromatography and analytical ultracentrifugation, and also binds to its putative receptor on naïve B cells and activated T cell subsets. Importantly, for the first time, we report the structure of BTNL2 as determined by solution NMR spectroscopy and also the picosecond-nanosecond timescale backbone dynamics of this domain. The N-terminal ectodomain of BTNL2, which was able to inhibit T cell function as well, exhibits distinctive structural features. The N-terminal ectodomain of BTNL2 has a significantly reduced surface area in the front sheet due to the non-canonical conformation of the CC' loop, which provides important insights into the recognition of its presently unknown binding partner.

The CC' loop of IgV domains of the immune checkpoint receptors, plays a key role in receptor:ligand affinity modulation.

Antibodies targeting negative regulators of immune checkpoints have shown unprecedented and durable response against variety of malignancies. While the concept of blocking the negative regulators of the immune checkpoints using mAbs appears to be an outstanding approach, their limited effect and several drawbacks, calls for the rational design of next generation of therapeutics. Soluble isoforms of the negative regulators of immune checkpoint pathways are expressed naturally and regulate immune responses. This suggests, affinity-modified versions of these self-molecules could be effective lead molecules for immunotherapy. To obtain better insights on the hotspot regions for modification, we have analysed structures of 18 immune receptor:ligand complexes containing the IgV domain. Interestingly, this analysis reveals that the CC' loop of IgV domain, a loop which is distinct from CDRs of antibodies, plays a pivotal role in affinity modulation, which was previously not highlighted. It is noteworthy that a ~5-residue long CC' loop in a ~120 residue protein makes significant number of hydrophobic and polar interactions with its cognate ligand. The post-interaction movement of CC' loop to accommodate the incoming ligands, seems to provide additional affinity to the interactions. In silico replacement of the CC' loop of TIGIT with that of Nectin-2 and PVR followed by protein docking trials suggests a key role of the CC' loop in affinity modulation in the TIGIT/Nectin pathway. The CC' loop appears to be a hotspot for the affinity modification without affecting the specificity to their cognate receptors.

The big Cs of the informed consent form: compliance and comprehension.

The informed consent process is a shield which protects subjects from harms that may be caused by a scientific enquiry. Only a competent participant with a complete understanding of the trial can give informed consent. Although the content of a valid informed consent form has been established, the Drugs and Cosmetics (First Amendment) Rules, 2013 have stipulated that ICFs must fulfil the requirements of Appendix V of Schedule Y. We considered 50 ICFs and analysed whether they complied with Appendix V. Our analysis reveals a gloomy picture, with 70% of the ICFs deviating from the requirements of the law. We have identified the elements most commonly overlooked in the ICFs analysed. We recommend certain points which must be incorporated into ICFs to help participants better understand the trial. Our findings indicate that adequate action needs to be taken to ensure the protection of the rights of research participants.