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Sci. Signal., 23 February 2010
Vol. 3, Issue 110, p. ec62
[DOI: 10.1126/scisignal.3110ec62]

EDITORS' CHOICE

Biochemistry Quantifying Interactions

Nancy R. Gough

Science Signaling, AAAS, Washington, DC 20005, USA

Two groups used optical fluorescence techniques to evaluate the kinetics of protein-protein interactions and produce results that challenge defining principles in the interaction of T cell receptors (TCRs) with peptide-bound major histocompatibility complexes (pMHCs) (Huppa et al.) and calcium channels with calmodulin (Liu et al.).

TCRs function as single-molecule sensors, allowing T cells to respond to very low concentrations of antigen in the context of pMHCs. Huppa et al. devised a method to analyze single-molecule fluorescence resonance energy transfer (smFRET) of TCRs present on T cells and pMHCs embedded in a planar lipid bilayer. They found that in the context of the immunological synapse, the affinity of the TCR-pMHC interaction was significantly increased compared to that in solution with surface plasmon resonance. Whereas the dissociation rate was increased (on the order of 10-fold) compared with measurements made in solution for this interaction, the association rate was markedly increased (on the order of 100-fold) compared with measurements made in solution (thus producing the increase in affinity). The difference between dissociation rates measured in solution versus those measured by Huppa et al. was lost if the actin cytoskeleton was disrupted by treatment of the T cells with latrunculin A or cytochalasin D, suggesting that cytoskeletal dynamics destabilize the TCR-pMHC interaction. Unexpectedly, blocking the coreceptor CD4, which is thought to bind to pMHC with the TCR, did not alter the affinity of the TCR-pMHC interaction, which indicates that CD4 functions to reinforce downstream signaling events and not to promote the interaction between the TCR and its ligand.

In a second study, Liu et al. provide evidence for a nonconstitutive association between the calcium-binding protein calmodulin (CaM) and long splice variants of two calcium channels (Cav1.3 and Cav1.4) using a combination of electrophysiology and FRET. This work was motivated by a mutation that causes night blindness in which the Cav1.4 channel prematurely terminates and is missing its distal carboxyl tail (DCT), resulting in a Ca2+-dependent inactivation that is not present in the full-length protein. With a FRET two-hybrid assay, Liu et al. found that the module that inhibits Ca2+-dependent inactivation (ICDI) bound to the CaM-binding region (IQ) of the Cav1.3 channel and could compete with calcium-free CaM (apoCaM) for binding to the IQ domain. With a FRET-based optical sensor for apoCaM, Liu et al. measured the concentration of apoCaM and Ca2+-dependent inactivation in the same cells and found that the ICDI present in long forms of human Cav1.3 and Cav1.4 is a competitive inhibitor of apoCaM, thereby preventing Ca2+-dependent inactivation. These results suggest that there are two mechanisms by which calcium regulation of calcium channels may occur: (i) through a constitutive, high-affinity interaction with apoCaM that enables changes in calcium concentration to directly regulate channel activity in short forms of the channels lacking the ICDI domain and (ii) by changes in the abundance of apoCaM, which acts as a competitive inhibitor of the binding of apoCaM to channels containing the ICDI domain. This latter scenario would enable variations in the abundance of CaM or proteins that buffer the concentration of apoCaM to influence channel activity.

J. B. Huppa, M. Axmann, M. A. Mörtelmaier, B. F. Lillemeier, E. W. Newell, M. Brameshuber, L. O. Klein, G. J. Schütz, M. M. Davis, TCR-peptide-MHC interactions in situ show accelerated kinetics and increased affinity. Nature 463, 963–967 (2010). [PubMed]

X. Liu, P. S. Yang, W. Yang, D. T. Yue, Enzyme-inhibitor-like tuning of Ca2+ channel connectivity with calmodulin. Nature 463, 968–972 (2010). [PubMed]

Citation: N. R. Gough, Quantifying Interactions. Sci. Signal. 3, ec62 (2010).



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