Dibyendu Samanta

Protein Folding by Domain V of Escherichia coli 23S rRNA: Specificity of RNA-Protein Interactions

The peptidyl transferase center, present in domain V of 23S rRNA of eubacteria and large rRNA of plants and animals, can act as a general protein folding modulator. Here we show that a few specific nucleotides in Escherichia coli domain V RNA bind to unfolded proteins and, as shown previously, bring the trapped proteins to a folding-competent state before releasing them. These nucleotides are the same for the proteins studied so far: bovine carbonic anhydrase, lactate dehydrogenase, malate dehydrogenase, and chicken egg white lysozyme. The amino acids that interact with these nucleotides are also found to be specific in the two cases tested: bovine carbonic anhydrase and lysozyme. They are either neutral or positively charged and are present in random coils on the surface of the crystal structure of both the proteins. In fact, two of these amino acid-nucleotide pairs are identical in the two cases. How these features might help the process of protein folding is discussed.

Role of the ribosome in protein folding

In all organisms, the ribosome synthesizes and folds full length polypeptide chains into active three-dimensional conformations. The nascent protein goes through two major interactions, first with the ribosome which synthesizes the polypeptide chain and holds it for a considerable length of time, and then with the chaperones. Some of the chaperones are found in solution as well as associated to the ribosome. A number of in vitro and in vivo experiments revealed that the nascent protein folds through specific interactions of some amino acids with the nucleotides in the peptidyl transferase center (PTC) in the large ribosomal subunit. The mechanism of this folding differs from self-folding. In this article, we highlight the folding of nascent proteins on the ribosome and the influence of chaperones etc. on protein folding.

Structure of Nectin-2 reveals determinants of homophilic and heterophilic interactions that control cell–cell adhesion

Nectins are members of the Ig superfamily that mediate cell–cell adhesion through homophilic and heterophilic interactions. We have determined the crystal structure of the nectin-2 homodimer at 1.3 Å resolution. Structural analysis and complementary mutagenesis studies reveal the basis for recognition and selectivity among the nectin family members. Notably, the close proximity of charged residues at the dimer interface is a major determinant of the binding affinities associated with homophilic and heterophilic interactions within the nectin family. Our structural and biochemical data provide a mechanistic basis to explain stronger heterophilic versus weaker homophilic interactions among these family members and also offer insights into nectin-mediated transinteractions between engaging cells.

Nectin family of cell-adhesion molecules: structural and molecular aspects of function and specificity.

Cell–cell adhesive processes are central to the physiology of multicellular organisms. A number of cell surface molecules contribute to cell–cell adhesion, and the dysfunction of adhesive processes underlies numerous developmental defects and inherited diseases. The nectins, a family of four immunoglobulin superfamily members (nectin-1 to -4), interact through their extracellular domains to support cell–cell adhesion. While both homophilic and heterophilic interactions among the nectins are implicated in cell–cell adhesion, cell-based and biochemical studies suggest heterophilic interactions are stronger than homophilic interactions and control a range of physiological processes. In addition to interactions within the nectin family, heterophilic associations with nectin-like molecules, immune receptors, and viral glycoproteins support a wide range of biological functions, including immune modulation, cancer progression, host-pathogen interactions and immune evasion. We review current structural and molecular knowledge of nectin recognition processes, with a focus on the biochemical and biophysical determinants of affinity and selectivity that drive distinct nectin associations. These proteins and interactions are discussed as potential targets for immunotherapy.

Involvement of mitochondrial ribosomal proteins in ribosomal RNA mediated protein folding

The peptidyl transferase center of the domain V of large ribosomal RNA in the prokaryotic and eukaryotic cytosolic ribosomes acts as general protein folding modulator. We showed earlier that one part of the domain V (RNA1 containing the peptidyl transferase loop) binds unfolded protein and directs it to a folding competent state (FCS) that is released by the other part (RNA2) to attain the folded native state by itself. Here we show that the peptidyl transferase loop of the mitochondrial ribosome releases unfolded proteins in FCS extremely slowly despite its lack of the rRNA segment analogous to RNA2. The release of FCS can be hastened by the equivalent activity of RNA2 or the large subunit proteins of the mitochondrial ribosome. The RNA2 or large subunit proteins probably introduce some allosteric change in the peptidyl transferase loop to enable it to release proteins in FCS.

Compensatory Mechanisms Allow Undersized Anchor-deficient Class I MHC Ligands to Mediate Pathogenic Autoreactive T Cell Responses

Self-reactive T cells must escape thymic negative selection to mediate pathogenic autoimmunity. In the NOD mouse model of autoimmune diabetes, several β cell–cytotoxic CD8 T cell populations are known, with the most aggressive of these represented by AI4, a T cell clone with promiscuous Ag-recognition characteristics. We identified a long-elusive β cell–specific ligand for AI4 as an unusually short H-2Db–binding 7-mer peptide lacking a C-terminal anchor residue and derived from the insulin A chain (InsA14–20). Crystallography reveals that compensatory mechanisms permit peptides lacking a C-terminal anchor to bind sufficiently to the MHC to enable destructive T cell responses, yet allow cognate T cells to avoid negative selection. InsA14–20 shares two solvent-exposed residues with previously identified AI4 ligands, providing a structural explanation for AI4’s promiscuity. Detection of AI4-like T cells, using mimotopes of InsA14–20 with improved H-2Db–binding characteristics, establishes the AI4-like T cell population as a consistent feature of the islet infiltrates of NOD mice. Our work establishes undersized peptides as previously unrecognized targets of autoreactive CD8 T cells and presents a strategy for their further exploration as Ags in autoimmune disease.

Structural and functional characterization of a single-chain peptide–MHC molecule that modulates both naive and activated CD8+ T cells

Peptide–MHC (pMHC) multimers, in addition to being tools for tracking and quantifying antigen-specific T cells, can mediate downstream signaling after T-cell receptor engagement. In the absence of costimulation, this can lead to anergy or apoptosis of cognate T cells, a property that could be exploited in the setting of autoimmune disease. Most studies with class I pMHC multimers used noncovalently linked peptides, which can allow unwanted CD8+ T-cell activation as a result of peptide transfer to cellular MHC molecules. To circumvent this problem, and given the role of self-reactive CD8+ T cells in the development of type 1 diabetes, we designed a single-chain pMHC complex (scKd.IGRP) by using the class I MHC molecule H-2Kd and a covalently linked peptide derived from islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP206–214), a well established autoantigen in NOD mice. X-ray diffraction studies revealed that the peptide is presented in the groove of the MHC molecule in canonical fashion, and it was also demonstrated that scKd.IGRP tetramers bound specifically to cognate CD8+ T cells. Tetramer binding induced death of naive T cells and in vitro- and in vivo-differentiated cytotoxic T lymphocytes, and tetramer-treated cytotoxic T lymphocytes showed a diminished IFN-γ response to antigen stimulation. Tetramer accessibility to disease-relevant T cells in vivo was also demonstrated. Our study suggests the potential of single-chain pMHC tetramers as possible therapeutic agents in autoimmune disease. Their ability to affect the fate of naive and activated CD8+ T cells makes them a potential intervention strategy in early and late stages of disease.

Structural, mutational and biophysical studies reveal a canonical mode of molecular recognition between immune receptor TIGIT and nectin-2

HighlightsT‐cell stimulatory and inhibitory signals control mammalian immune system.TIGIT:nectin‐2 interaction delivers inhibitory signals to T cell.This study reveals structural and biochemical basis of TIGIT:nectin‐2 recognition.A distinctive “lock‐and‐key” mechanism supports this molecular interaction.This study provides basis for rational manipulation of TIGIT:nectin2 interaction. ABSTRACT In addition to antigen‐specific stimulation of T cell receptor (TCR) by a peptide‐MHC complex, the functional outcome of TCR engagement is regulated by antigen‐independent costimulatory signals. Costimulatory signals are provided by an array of interactions involving activating and inhibitory receptors expressed on T cells and their cognate ligands on antigen presenting cells. T cell immunoglobulin and ITIM domain (TIGIT), a recently identified immune receptor expressed on T and NK cells, upon interaction with either of its two ligands, nectin‐2 or poliovirus receptor (PVR), inhibits activation of T and NK cells. Here we report the crystal structure of the human TIGIT ectodomain, which exhibits the classic two‐layer &bgr;‐sandwich topology observed in other immunoglobulin super family (IgSF) members. Biophysical studies indicate that TIGIT is monomeric in solution but can form a dimer at high concentrations, consistent with the observation of a canonical immunoglobulin‐like dimer interface in the crystalline state. Based on existing structural data, we present a model of the TIGIT:nectin‐2 complex and utilized complementary biochemical studies to map the nectin‐binding interface on TIGIT. Our data provide important structural and biochemical determinants responsible for the recognition of nectin‐2 by TIGIT. Defining the TIGIT:nectin‐2 binding interface provides the basis for rational manipulation of this molecular interaction for the development of immunotherapeutic reagents in autoimmunity and cancer.

Mechanism of ribosome assisted protein folding: a new insight into rRNA functions.

The peptidyl transferase center (PTC), present in the domain V of 23S rRNA of bacteria can act as a general protein folding modulator. Any general function of a nucleic acid polymer (DNA or RNA) is always related to specific sequence/sequences. The ribosome mediated protein folding also involves a specific interaction between the nucleotides of peptidyl transferase center and the amino acids of an unfolded protein. In this article the mechanism of rRNA assisted protein folding and its significance in the light of high resolution crystal structure of ribosome are discussed.