Biomedical subjects

Beenu Moza

Publications and source records attributed to Beenu Moza.

6 recordsLinked to original sources

PubMed2006

Molecular basis of TCR selectivity, cross-reactivity, and allelic discrimination by a bacterial superantigen: integrative functional and energetic mapping of the SpeC-Vbeta2.1 molecular interface.

Superantigens activate large fractions of T cells through unconventional interactions with both TCR beta-chain V domains (Vbetas) and MHC class II molecules. The bacterial superantigen streptococcal pyrogenic exotoxin C (SpeC) primarily stimulates human Vbeta2(+) T cells. Herein, we have analyzed the SpeC-Vbeta2.1 interaction by mutating all SpeC residues that make contact with Vbeta2.1 and have determined the energetic and functional consequences of these mutations. Our comprehensive approach, including mutagenesis, functional readouts from both bulk T cell populations, and an engineered Vbeta2.1(+) Jurkat T cell, as well as surface plasmon resonance binding analysis, has defined the SpeC "functional epitope" for TCR engagement. Although only two SpeC residues (Tyr(15) and Arg(181)) are critical for activation of virtually all human CD3(+) T cells, a larger cluster of four hot spot residues are required for interaction with Vbeta2.1. Three of these residues (Tyr(15), Phe(75), and Arg(181)) concentrate their binding energy on the CDR2 loop residue Ser(52a), a noncanonical residue insertion found only in Vbeta2 and Vbeta4 chains. Plasticity of this loop is important for recognition by SpeC. Although SpeC interacts with the Vbeta2.1 hypervariable CDR3 loop, our data indicate these contacts have little to no influence on the functional interaction with Vbeta2.1. These studies also provide a molecular basis for selectivity and cross-reactivity of SpeC-TCR recognition and reveal a degree of fine specificity in these interactions, whereby certain SpeC mutants are capable of distinguishing between different alleles of the same Vbeta domain subfamily.

Alleles↗

PubMed2006

Long-range cooperative binding effects in a T cell receptor variable domain.

Although cellular processes depend on protein-protein interactions, our understanding of molecular recognition between proteins remains far from comprehensive. Protein-protein interfaces are structural and energetic mosaics in which a subset of interfacial residues, called hot spots, contributes disproportionately to the affinity of the complex. These hot-spot residues can be further clustered into hot regions. It has been proposed that binding energetics between residues within a hot region are cooperative, whereas those between hot regions are strictly additive. If this idea held true for all protein-protein interactions, then energetically significant long-range conformational effects would be unlikely to occur. In the present study, we show cooperative binding energetics between distinct hot regions that are separated by >20 A. Using combinatorial mutagenesis and surface plasmon resonance binding analysis to dissect additivity and cooperativity in a complex formed between a variable domain of a T cell receptor and a bacterial superantigen, we find that combinations of mutations from each of two hot regions exhibited significant cooperative energetics. Their connecting sequence is composed primarily of a single beta-strand of the T cell receptor variable Ig domain, which has been observed to undergo a strand-switching event and does not form an integral part of the stabilizing core of this Ig domain. We propose that these cooperative effects are propagated through a dynamic structural network. Cooperativity between hot regions has significant implications for the prediction and inhibition of protein-protein interactions.

Bacterial Toxins↗

PubMed2006

A unique molten globule state occurs during unfolding of cytochrome c by LiClO4 near physiological pH and temperature: structural and thermodynamic characterization.

We have carried out denaturation studies of bovine cytochrome c (cyt c) by LiClO4 at pH 6.0 and 25 degrees C by observing changes in difference molar absorbance at 400 nm (Deltaepsilon400), mean residue ellipticities at 222 nm ([theta]222) and difference mean residue ellipticity at 409 nm (Delta[theta]409). The denaturation is a three-step process when measured by Deltaepsilon400 and Delta[theta]409, and it is a two-step process when monitored by [theta]222. The stable folding intermediate state has been characterized by near- and far-UV circular dichroism, tryptophan fluorescence, 8-anilino-1-naphthalene sulfonic acid (ANS) binding, and intrinsic viscosity measurements. A comparison of the conformational and thermodynamic properties of the LiClO4-induced molten globule (MG) state with those induced by other solvent conditions (e.g., low pH, LiCl, and CaCl2) suggests that LiClO4 induces a unique MG state, i.e., (i) the core in the LiClO4-induced state retains less secondary and tertiary structure than that in the MG states obtained in other solvent conditions, and (ii) the thermodynamic stability associated with the LiClO4-induced process, native state <--> MG state, is the same as that observed for each transition between native and MG states induced by other solvent conditions.

Animals↗

PubMed2005

Characterization of T cell receptors engineered for high affinity against toxic shock syndrome toxin-1.

Superantigens, including bacterial enterotoxins, are a family of proteins that bind simultaneously to MHC class II molecules and the Vbeta regions of T cell receptors. This cross-linking results in the activation of a large population of T cells that release massive amounts of inflammatory cytokines, ultimately causing a condition known as toxic shock syndrome. The staphylococcal superantigen toxic shock syndrome toxin-1 (TSST-1) is a causative agent of this disease, but its structure in complex with the cognate T cell receptor (human Vbeta2.1) has not been determined. To understand the molecular details of the interaction and to develop high affinity antagonists to TSST-1, we used directed evolution to generate a panel of high affinity receptors for TSST-1. Yeast display libraries of random and site-directed hVbeta2.1 mutants were selected for improved domain stability and for higher affinity binding to TSST-1. Stability mutations allowed the individual Vbeta domains to be expressed in a bacterial expression system. Affinity mutations were generated in CDR2 and FR3 residues, yielding improvements in affinity of greater than 10,000-fold (a K(D) value of 180 pmol). Alanine scanning mutagenesis of hVbeta2.1 wild-type and mutated residues allowed us to generate a map of the binding site for TSST-1 and to construct a docking model for the hVbeta2.1-TSST-1 complex. Our experiments suggest that the energetic importance of a single hVbeta2.1 wild-type residue likely accounts for the restriction of TSST-1 specificity to only this human Vbeta region. The high affinity mutants described here thus provide critical insight into the molecular basis of TSST-1 specificity and serve as potential leads toward the development of therapeutic agents for superantigen-mediated disease.

Alanine↗

PubMed2003

Equilibrium studies of the effect of difference in sequence homology on the mechanism of denaturation of bovine and horse cytochromes-c.

We have carried out equilibrium studies of the effect of the amino acid residue difference in the primary structure of bovine cytochrome-c (b-cyt-c) and horse cyt-c (h-cyt-c) on the mechanism of their folding <--> unfolding processes at pH 6.0 and 25 degrees C. It has been observed that guanidinium chloride (GdmCl)-induced denaturation of b-cyt-c follows a two-state mechanism and that of h-cyt-c is not a two-state process. This conclusion is reached from the coincidence and non-coincidence of GdmCl-induced transition curves of bovine and horse proteins, respectively, monitored by measurements of absorbance at 405, 530 and 695 nm and circular dichroism (CD) at 222, 416 and 405 nm. These measurements on h-cyt-c in the presence of GdmCl in the concentration range 0.75-2.0 M also suggest that the protein retains all the native far-UV CD but has slightly perturbed tertiary interaction. The intermediate in the presence of these low denaturant concentrations does not have the structural characteristics of a molten globule as judged by the 8-Anilino-1-napthalene sulfonic acid (ANS) binding and near-UV CD experiments. We have also carried out thermal denaturation studies of bovine and horse cyts-c in the presence of GdmCl monitored by absorbance at 405 nm and far-UV CD at 222 nm. The heat-induced denaturation measurements in the presence of the denaturant show (1) that denaturation of b-cyt-c is a two-state process and that of h-cyt-c does not follow a two-state mechanism, and (2) that the enthalpy change on denaturation of both proteins strongly depends on GdmCl concentration.

Anilino Naphthalenesulfonates↗

PubMed2003

Conformational and thermodynamic characterization of the molten globule state occurring during unfolding of cytochromes-c by weak salt denaturants.

The denaturation of bovine and horse cytochromes-c by weak salt denaturants (LiCl and CaCl(2)) was measured at 25 degrees C by observing changes in molar absorbance at 400 nm (Delta epsilon(400)) and circular dichroism (CD) at 222 and 409 nm. Measurements of Delta epsilon(400) and mean residue ellipticity at 409 nm ([theta](409)) gave a biphasic transition for both modes of denaturation of cytochromes-c. It has been observed that the first denaturation phase, N (native) conformation <--> X (intermediate) conformation and the second denaturation phase, X conformation <--> D (denatured) conformation are reversible. Conformational characterization of the X state by the far-UV CD, 8-anilino-1-naphthalene sulfonic acid (ANS) binding, and intrinsic viscosity measurements led us to conclude that the X state is a molten globule state. Analysis of denaturation transition curves for the stability of different states in terms of Gibbs energy change at pH 6.0 and 25 degrees C led us to conclude that the N state is more stable than the X state by 9.55 +/- 0.32 kcal mol(-1), whereas the X state is more stable than the D state by only 1.40 +/- 0.25 kcal mol(-1). We have also studied the effect of temperature on the equilibria, N conformation <--> X conformation and X conformation <--> D conformation in the presence of different denaturant concentrations using two different optical probes, namely, [theta](222) and Delta epsilon(400). These measurements yielded T(m), (midpoint of denaturation) and Delta H(m) (enthalpy change) at T(m) as a function of denaturant concentration. A plot of Delta H(m) versus corresponding T(m) was used to determine the constant-pressure heat capacity change, Delta C(p) (= ( partial differential Delta H(m)/ partial differential T(m))(p)). Values of Delta C(p) for N conformation <--> X conformation and X conformation <--> D conformation is 0.92 +/- 0.02 kcal mol(-1) K(-1) and 0.41 +/- 0.01 kcal mol(-1) K(-1), respectively. These measurements suggested that about 30% of the hydrophobic groups in the molten globule state are not accessible to the water.

Animals↗