A new twist in TCR diversity revealed by a forbidden alphabeta TCR.

We report crystal structures of a negatively selected T cell receptor (TCR) that recognizes two I-A(u)-restricted myelin basic protein peptides and one of its peptide/major histocompatibility complex (pMHC) ligands. Unusual complementarity-determining region (CDR) structural features revealed by our analyses identify a previously unrecognized mechanism by which the highly variable CDR3 regions define ligand specificity. In addition to the pMHC contact residues contributed by CDR3, the CDR3 residues buried deep within the V alpha/V beta interface exert indirect effects on recognition by influencing the V alpha/V beta interdomain angle. This phenomenon represents an additional mechanism for increasing the potential diversity of the TCR repertoire. Both the direct and indirect effects exerted by CDR residues can impact global TCR/MHC docking. Analysis of the available TCR structures in light of these results highlights the significance of the V alpha/V beta interdomain angle in determining specificity and indicates that TCR/pMHC interface features do not distinguish autoimmune from non-autoimmune class II-restricted TCRs.

Ca2+ indicators based on computationally redesigned calmodulin-peptide pairs.

The binding interface of calmodulin and a calmodulin binding peptide were reengineered by computationally designing complementary bumps and holes. This redesign led to the development of sensitive and specific pairs of mutant proteins used to sense Ca(2+) in a second generation of genetically encoded Ca(2+) indicators (cameleons). These cameleons are no longer perturbed by large excesses of native calmodulin, and they display Ca(2+) sensitivities tuned over a 100-fold range (0.6-160 microM). Incorporation of circularly permuted Venus in place of Citrine results in a 3- to 5-fold increase in the dynamic range. These redesigned cameleons show significant improvements over previous versions in the ability to monitor Ca(2+) in the cytoplasm as well as distinct subcellular localizations, such as the plasma membrane of neurons and the mitochondria.

Rational design of intercellular adhesion molecule-1 (ICAM-1) variants for antagonizing integrin lymphocyte function-associated antigen-1-dependent adhesion.

The interaction between integrin lymphocyte function-associated antigen-1 (LFA-1) and its ligand intercellular adhesion molecule-1 (ICAM-1) is critical in immunological and inflammatory reactions but, like other adhesive interactions, is of low affinity. Here, multiple rational design methods were used to engineer ICAM-1 mutants with enhanced affinity for LFA-1. Five amino acid substitutions 1) enhance the hydrophobicity and packing of residues surrounding Glu-34 of ICAM-1, which coordinates to a Mg2+ in the LFA-1 I domain, and 2) alter associations at the edges of the binding interface. The affinity of the most improved ICAM-1 mutant for intermediate- and high-affinity LFA-1 I domains was increased by 19-fold and 22-fold, respectively, relative to wild type. Moreover, potency was similarly enhanced for inhibition of LFA-1-dependent ligand binding and cell adhesion. Thus, rational design can be used to engineer novel adhesion molecules with high monomeric affinity; furthermore, the ICAM-1 mutant holds promise for targeting LFA-1-ICAM-1 interaction for biological studies and therapeutic purposes.

Close agreement between the orientation dependence of hydrogen bonds observed in protein structures and quantum mechanical calculations.

Hydrogen bonding is a key contributor to the exquisite specificity of the interactions within and between biological macromolecules, and hence accurate modeling of such interactions requires an accurate description of hydrogen bonding energetics. Here we investigate the orientation and distance dependence of hydrogen bonding energetics by combining two quite disparate but complementary approaches: quantum mechanical electronic structure calculations and protein structural analysis. We find a remarkable agreement between the energy landscapes obtained from the electronic structure calculations and the distributions of hydrogen bond geometries observed in protein structures. In contrast, molecular mechanics force fields commonly used for biomolecular simulations do not consistently exhibit close correspondence to either quantum mechanical calculations or experimentally observed hydrogen bonding geometries. These results suggest a route to improved energy functions for biological macromolecules that combines the generality of quantum mechanical electronic structure calculations with the accurate context dependence implicit in protein structural analysis.