The Structure of Ca2+ Sensor Case16 Reveals the Mechanism of Reaction to Low Ca2+ Concentrations

Sensors

Lukas Leder¹  Wilhelm Stark¹  Felix Freuler²  May Marsh²  Marco Meyerhofer²  Thomas Stettler²  Lorenz M. Mayr²  Olga V. Britanova³  Lydia A. Strukova³  Dmitriy M. Chudakov³ 

[1] Novartis Pharma AG, NIBR/CPC/LFP, Lichtstrasse 35, CH-4002 Basel, Switzerland; E-Mails:;Shemiakin-Ovchinnikov Institute of Bioorganic Chemistry, RAS, Miklukho-Maklaya str. 16/10, 117997, Moscow, Russia; E-Mails:

关键词: circularly permuted green fluorescent protein;    genetically encoded;    fluorescent calcium indicator protein;    crystal structure;    calcium sensor;   

DOI  :  10.3390/s100908143

来源: mdpi

【 摘 要 】

Here we report the first crystal structure of a high-contrast genetically encoded circularly permuted green fluorescent protein (cpGFP)-based Ca²⁺ sensor, Case16, in the presence of a low Ca²⁺ concentration. The structure reveals the positioning of the chromophore within Case16 at the first stage of the Ca²⁺-dependent response when only two out of four Ca²⁺-binding pockets of calmodulin (CaM) are occupied with Ca²⁺ ions. In such a “half Ca²⁺-bound state”, Case16 is characterized by an incomplete interaction between its CaM-/M13-domains. We also report the crystal structure of the related Ca²⁺ sensor Case12 at saturating Ca²⁺ concentration. Based on this structure, we postulate that cpGFP-based Ca²⁺ sensors can form non-functional homodimers where the CaM-domain of one sensor molecule binds symmetrically to the M13-peptide of the partner sensor molecule. Case12 and Case16 behavior upon addition of high concentrations of free CaM or M13-peptide reveals that the latter effectively blocks the fluorescent response of the sensor. We speculate that the demonstrated intermolecular interaction with endogenous substrates and homodimerization can impede proper functioning of this type of Ca²⁺ sensors in living cells.

【 授权许可】

CC BY   
© 2010 by the authors; licensee MDPI, Basel, Switzerland.