Biomedical subjects

Jennifer N Wingard

Publications and source records attributed to Jennifer N Wingard.

3 recordsLinked to original sources

PubMed2006

Structural basis for calcium-induced inhibition of rhodopsin kinase by recoverin.

Recoverin, a member of the neuronal calcium sensor branch of the EF-hand superfamily, serves as a calcium sensor that regulates rhodopsin kinase (RK) activity in retinal rod cells. We report here the NMR structure of Ca(2+)-bound recoverin bound to a functional N-terminal fragment of rhodopsin kinase (residues 1-25, called RK25). The overall main-chain structure of recoverin in the complex is similar to structures of Ca(2+)-bound recoverin in the absence of target (<1.8A root-mean-square deviation). The first eight residues of recoverin at the N terminus are solvent-exposed, enabling the N-terminal myristoyl group to interact with target membranes, and Ca(2+) is bound at the second and third EF-hands of the protein. RK25 in the complex forms an amphipathic helix (residues 4-16). The hydrophobic face of the RK25 helix (Val-9, Val-10, Ala-11, Ala-14, and Phe-15) interacts with an exposed hydrophobic groove on the surface of recoverin lined by side-chain atoms of Trp-31, Phe-35, Phe-49, Ile-52, Tyr-53, Phe-56, Phe-57, Tyr-86, and Leu-90. Residues of recoverin that contact RK25 are highly conserved, suggesting a similar target binding site structure in all neuronal calcium sensor proteins. Site-specific mutagenesis and deletion analysis confirm that the hydrophobic residues at the interface are necessary and sufficient for binding. The recoverin-RK25 complex exhibits Ca(2+)-induced binding to rhodopsin immobilized on concanavalin-A resin. We propose that Ca(2+)-bound recoverin is bound between rhodopsin and RK in a ternary complex on rod outer segment disk membranes, thereby blocking RK interaction with rhodopsin at high Ca(2+).

Amino Acid Sequence↗

PubMed2005

A flagellum-specific calcium sensor.

The flagellar calcium-binding protein (FCaBP) of the flagellated protozoan Trypanosoma cruzi associates with the flagellar membrane via its N-terminal myristate and palmitate moieties in a calcium-modulated, conformation-dependent manner. This mechanism of localization is similar to that described for neuronal calcium sensors, which undergo calcium-dependent changes in conformation, which modulate the availability of the acyl groups for membrane interaction and partner association. To test whether FCaBP undergoes a calcium-dependent conformational change and to explore the role of such a change in flagellar targeting, we first introduced point mutations into each of the two EF-hand calcium-binding sites of FCaBP to define their affinities. Analysis of recombinant EF-3 mutant (E151Q), EF-4 mutant (E188Q), and double mutant proteins showed EF-3 to be the high affinity site (Kd approximately 9 microM) and EF-4 the low affinity site (Kd approximately 120 microM). These assignments also correlated with partial (E188Q), nearly complete (E151Q), and complete (E151Q,E188Q) disruption of calcium-induced conformational changes determined by NMR spectrometry. We next expressed the FCaBP E151Q mutant and the double mutant in T. cruzi epimastigotes. These transproteins localized to the flagellum, suggesting the existence of a calcium-dependent interaction of FCaBP that is independent of its intrinsic calcium binding capacity. Several proteins were identified by FCaBP affinity chromatography that interact with FCaBP in a calcium-dependent manner, but with differential dependence on calcium-binding by FCaBP. These findings may have broader implications for the calcium acyl switch mechanism of protein regulation.

Animals↗

PubMed2005

Structural analysis of Mg2+ and Ca2+ binding to CaBP1, a neuron-specific regulator of calcium channels.

CaBP1 (calcium-binding protein 1) is a 19.4-kDa protein of the EF-hand superfamily that modulates the activity of Ca(2+) channels in the brain and retina. Here we present data from NMR, microcalorimetry, and other biophysical studies that characterize Ca(2+) binding, Mg(2+) binding, and structural properties of recombinant CaBP1 purified from Escherichia coli. Mg(2+) binds constitutively to CaBP1 at EF-1 with an apparent dissociation constant (K(d)) of 300 microm. Mg(2+) binding to CaBP1 is enthalpic (DeltaH = -3.725 kcal/mol) and promotes NMR spectral changes, indicative of a concerted Mg(2+)-induced conformational change. Ca(2+) binding to CaBP1 induces NMR spectral changes assigned to residues in EF-3 and EF-4, indicating localized Ca(2+)-induced conformational changes at these sites. Ca(2+) binds cooperatively to CaBP1 at EF-3 and EF-4 with an apparent K(d) of 2.5 microM and a Hill coefficient of 1.3. Ca(2+) binds to EF-1 with low affinity (K(d) >100 microM), and no Ca(2+) binding was detected at EF-2. In the absence of Mg(2+) and Ca(2+), CaBP1 forms a flexible molten globule-like structure. Mg(2+) and Ca(2+) induce distinct conformational changes resulting in protein dimerization and markedly increased folding stability. The unfolding temperatures are 53, 74, and 76 degrees C for apo-, Mg(2+)-bound, and Ca(2+)-bound CaBP1, respectively. Together, our results suggest that CaBP1 switches between structurally distinct Mg(2+)-bound and Ca(2+)-bound states in response to Ca(2+) signaling. Both conformational states may serve to modulate the activity of Ca(2+) channel targets.

Amino Acid Sequence↗