Deepshikha Datta

Mechanism for antibody catalysis of the oxidation of water by singlet dioxygen

Wentworth et al. [Wentworth, P., Jones, L. H., Wentworth, A. D., Zhu, X. Y., Larsen, N. A., Wilson, I. A., Xu, X., Goddard, W. A., Janda, K. D., Eschenmoser, A. & Lerner, R. A. (2001) Science 293, 1806–1811] recently reported the surprising result that antibodies and T cell receptors efficiently catalyze the conversion of molecular singlet oxygen (1O2) plus water to hydrogen peroxide (HOOH). Recently, quantum mechanical calculations were used to delineate a plausible mechanism, involving reaction of 1O2 with two waters to form HOOOH (plus H2O), followed by formation of HOOOH dimer, which rearranges to form HOO—HOOO + H2O, which rearranges to form two HOOH plus 1O2 or 3O2. For a system with 18O H2O, this mechanism leads to a 2.2:1 ratio of 16O:18O in the product HOOH, in good agreement with the ratio 2.2:1 observed in isotope experiments by Wentworth et al. In this paper we use docking and molecular dynamics techniques (HierDock) to search various protein structures for sites that stabilize these products and intermediates predicted from quantum mechanical calculations. We find that the reaction intermediates for production of HOOH from 1O2 are stabilized at the interface of light and heavy chains of antibodies and T cell receptors. This inter Greek key domain interface structure is unique to antibodies and T cell receptors, but is not present in β2-microglobulin, which does not show any stabilization in our docking studies. This result is consistent with the experimentally observed lack of HOOH production in this system. Our results provide a plausible mechanism for the reactions and provide an explanation of the specific structural character of antibodies responsible for this unexpected chemistry.

Interaction of E. coli Outer-Membrane Protein A with Sugars on the Receptors of the Brain Microvascular Endothelial Cells

Esherichia coli, the most common gram‐negative bacteria, can penetrate the brain microvascular endothelial cells (BMECs) during the neonatal period to cause meningitis with significant morbidity and mortality. Experimental studies have shown that outer‐membrane protein A (OmpA) of E. coli plays a key role in the initial steps of the invasion process by binding to specific sugar moieties present on the glycoproteins of BMEC. These experiments also show that polymers of chitobiose (GlcNAcβ1‐4GlcNAc) block the invasion, while epitopes substituted with the L‐fucosyl group do not. We used HierDock computational technique that consists of a hierarchy of coarse grain docking method with molecular dynamics (MD) to predict the binding sites and energies of interactions of GlcNAcβ1‐4GlcNAc and other sugars with OmpA. The results suggest two important binding sites for the interaction of carbohydrate epitopes of BMEC glycoproteins to OmpA. We identify one site as the binding pocket for chitobiose (GlcNAcβ1‐4GlcNAc) in OmpA, while the second region (including loops 1 and 2) may be important for recognition of specific sugars. We find that the site involving loops 1 and 2 has relative binding energies that correlate well with experimental observations. This theoretical study elucidates the interaction sites of chitobiose with OmpA and the binding site predictions made in this article are testable either by mutation studies or invasion assays. These results can be further extended in suggesting possible peptide antagonists and drug design for therapeutic strategies. Proteins 2003;50:213–221.

Evaluation of the energetic contribution of an ionic network to beta-sheet stability.

We have evaluated the interaction energy of a three‐residue ionic network constructed on the β‐sheet surface of protein G using double mutant cycles. Although the two individual ion pairs were each stabilizing by ∼0.6 kcal/mol, the excess gain in stability for the triad was small (0.06 kcal/mol).

Selectivity and specificity of substrate binding in methionyl-tRNA synthetase

The accuracy of in vivo incorporation of amino acids during protein biosynthesis is controlled to a significant extent by aminoacyl‐tRNA synthetases (aaRS). This paper describes the application of the HierDock computational method to study the molecular basis of amino acid binding to the Escherichia coli methionyl tRNA synthetase (MetRS). Starting with the protein structure from the MetRS cocrystal, the HierDock calculations predict the binding site of methionine in MetRS to a root mean square deviation in coordinates (CRMS) of 0.55 Å for all the atoms, compared with the crystal structure. The MetRS conformation in the cocrystal structure shows good discrimination between cognate and the 19 noncognate amino acids. In addition, the calculated binding energies of a set of five methionine analogs show a good correlation (R2 = 0.86) to the relative free energies of binding derived from the measured in vitro kinetic parameters, Km and kcat. Starting with the crystal structure of MetRS without the methionine (apo‐MetRS), the putative binding site of methionine was predicted. We demonstrate that even the apo‐MetRS structure shows a preference for binding methionine compared with the 19 other natural amino acids. On comparing the calculated binding energies of the 20 natural amino acids for apo‐MetRS with those for the cocrystal structure, we observe that the discrimination against the noncognate substrate increases dramatically in the second step of the physical binding process associated with the conformation change in the protein.

A designed apoplastocyanin variant that shows reversible folding

Plastocyanin, like many other metalloproteins, does not undergo reversible folding, which is thought to be due to an irreversible conformational change in the copper-binding site. Moreover, apoplastocyanins ability to adopt a native tertiary structure is highly salt-dependent, and even in high salt, it has an irreversible thermal denaturation. Here, we report a designed apoplastocyanin variant, PCV, that is well folded and has reversible folding in both high and low salt conditions. This variant provides a tractable model for understanding and designing protein beta-sheets.