Coordination to lanthanide ions distorts binding site conformation in calmodulin

Abstract

<p id="d4574658e212">Calmodulin is essential to life in all eukaryotic cells and serves as a popular model for ion binding and activation in proteins. Calmodulin transduces complex calcium signals and acts on hundreds of effector proteins, but the sensitivity and complexity of this process make it difficult to characterize. Much work uses lanthanides as luminescent calcium substitutes to study ion binding and activation in calmodulin and other proteins. Using ultrafast 2D IR spectroscopy, we show that lanthanide ions perturb the finely tuned structure and dynamics of calmodulin’s binding sites. The temporal and spatial resolution of our measurements opens a new window into the study of protein−ion binding and demonstrates that seemingly innocuous ligand substitutions can significantly alter protein conformation. </p><p class="first" id="d4574658e215">The Ca ²⁺-sensing protein calmodulin (CaM) is a popular model of biological ion binding since it is both experimentally tractable and essential to survival in all eukaryotic cells. CaM modulates hundreds of target proteins and is sensitive to complex patterns of Ca ²⁺ exposure, indicating that it functions as a sophisticated dynamic transducer rather than a simple on/off switch. Many details of this transduction function are not well understood. Fourier transform infrared (FTIR) spectroscopy, ultrafast 2D infrared (2D IR) spectroscopy, and electronic structure calculations were used to probe interactions between bound metal ions (Ca ²⁺ and several trivalent lanthanide ions) and the carboxylate groups in CaM’s EF-hand ion-coordinating sites. Since Tb ³⁺ is commonly used as a luminescent Ca ²⁺ analog in studies of protein−ion binding, it is important to characterize distinctions between the coordination of Ca ²⁺ and the lanthanides in CaM. Although functional assays indicate that Tb ³⁺ fully activates many Ca ²⁺-dependent proteins, our FTIR spectra indicate that Tb ³⁺, La ³⁺, and Lu ³⁺ disrupt the bidentate coordination geometry characteristic of the CaM binding sites’ strongly conserved position 12 glutamate residue. The 2D IR spectra indicate that, relative to the Ca ²⁺-bound form, lanthanide-bound CaM exhibits greater conformational flexibility and larger structural fluctuations within its binding sites. Time-dependent 2D IR lineshapes indicate that binding sites in Ca ²⁺−CaM occupy well-defined configurations, whereas binding sites in lanthanide-bound-CaM are more disordered. Overall, the results show that binding to lanthanide ions significantly alters the conformation and dynamics of CaM’s binding sites. </p>