Intra- and Interdomain Effects Due to Mutation of Calcium-binding Sites in Calmodulin

Xiong, Liangwen; Kleerekoper, Quinn; Wang, Xu; Putkey, John A.

doi:10.1074/jbc.m109.065243

Cited by 24 publications

(38 citation statements)

References 54 publications

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“…A decreased resistance coefficient was also observed in the Phe19Ala mutant, demonstrating that the nature of the side-chain is an important factor in determining the mobility of the residue's backbone. The most profound effect on the resistance coefficient of Phe 19, however, was observed for replacement by alanine of Asp 20, a highly conserved residue at position 1 of the EF-1 Ca 2+ -binding loop (Supplementary Figure S1) [25], [35], essentially reversing the stabilizing effect of Ca 2+ binding. The surface exposed Phe 19 is located in an unstructured region at the beginning of the Ca 2+ -binding loop, and Ca 2+ coordination mediated by the adjacent Asp 20 may offer a stabilizing effect on the dynamics of the Phe 19 backbone.…”

Section: Resultsmentioning

confidence: 99%

Surface Dynamics in Allosteric Regulation of Protein-Protein Interactions: Modulation of Calmodulin Functions by Ca2+

2013

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Knowledge of the structural basis of protein-protein interactions (PPI) is of fundamental importance for understanding the organization and functioning of biological networks and advancing the design of therapeutics which target PPI. Allosteric modulators play an important role in regulating such interactions by binding at site(s) orthogonal to the complex interface and altering the protein's propensity for complex formation. In this work, we apply an approach recently developed by us for analyzing protein surfaces based on steered molecular dynamics simulation (SMD) to the study of the dynamic properties of functionally distinct conformations of a model protein, calmodulin (CaM), whose ability to interact with target proteins is regulated by the presence of the allosteric modulator Ca2+. Calmodulin is a regulatory protein that acts as an intracellular Ca2+ sensor to control a wide variety of cellular processes. We demonstrate that SMD analysis is capable of pinpointing CaM surfaces implicated in the recognition of both the allosteric modulator Ca2+ and target proteins. Our analysis of changes in the dynamic properties of the CaM backbone elicited by Ca2+ binding yielded new insights into the molecular mechanism of allosteric regulation of CaM-target interactions.

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Section: Resultsmentioning

confidence: 99%

Surface Dynamics in Allosteric Regulation of Protein-Protein Interactions: Modulation of Calmodulin Functions by Ca2+

2013

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“…While fast backbone dynamics contribute principally to Ca 2þ affinity, interactions also exist between lobes that influence Ca 2þ -binding properties (53)(54)(55). This raises the possibility that although ficoll-70, dextran-10, and sucrose are uncharged and produce no detectable effects on secondary structure, modulation of interlobe dynamics may influence Ca 2þ affinity.…”

Section: Volume Exclusion Conditions Do Not Modulate Ca 2d Affinity Fmentioning

confidence: 94%

Relative Cosolute Size Influences the Kinetics of Protein-Protein Interactions

Hoffman¹,

Wang²,

Sanabria

et al. 2015

Biophysical Journal

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Protein signaling occurs in crowded intracellular environments, and while high concentrations of macromolecules are postulated to modulate protein-protein interactions, analysis of their impact at each step of the reaction pathway has not been systematically addressed. Potential cosolute-induced alterations in target association are particularly important for a signaling molecule like calmodulin (CaM), where competition among >300 targets governs which pathways are selectively activated. To explore how high concentrations of cosolutes influence CaM-target affinity and kinetics, we methodically investigated each step of the CaM-target binding mechanism under crowded or osmolyte-rich environments mimicked by ficoll-70, dextran-10, and sucrose. All cosolutes stabilized compact conformers of CaM and modulated association kinetics by affecting diffusion and rates of conformational change; however, the results showed that differently sized molecules had variable effects to enhance or impede unique steps of the association pathway. On- and off-rates were modulated by all cosolutes in a compensatory fashion, producing little change in steady-state affinity. From this work insights were gained on how high concentrations of inert crowding agents and osmolytes fit into a kinetic framework to describe protein-protein interactions relevant for cellular signaling.

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“…Equilibrium Ca 2ϩ binding constants for CaM in the presence or absence of PEP-19 derivatives were determined using tyrosine fluorescence at 23°C as described previously (17). Data were collected with a QuantaMaster fluorimeter (Photo Technology International).…”

Section: Methodsmentioning

confidence: 99%