CALX-CBD1 Ca2+-Binding Cooperativity Studied by NMR Spectroscopy and ITC with Bayesian Statistics

Author(s)

Cardoso, Marcus V. C. ^{[1, 2]} ; Rivera, Jose D. ^[1] ; Vitale, Phelipe A. M. ^[1] ; Degenhardt, Maximilia F. S. ^[3] ; Abiko, Layara A. ^{[1, 4]} ; Oliveira, Cristiano L. P. ^[3] ; Salinas, Roberto K. ^[1]

Affiliation

^[1] Univ Sao Paulo, Inst Chem, Dept Biochem, Sao Paulo - Brazil

^[2] Univ Fed Ouro Preto, Dept Chem, Ouro Preto - Brazil

^[3] Univ Sao Paulo, Dept Expt Phys, Inst Phys, Sao Paulo - Brazil

^[4] Univ Basel, Biozentrum, Struct Biol & Biophys, Basel - Switzerland

• Document type: Journal article
• Source: BIOPHYSICAL JOURNAL; v. 119, n. 2, p. 337-348, JUL 21 2020.
• Web of Science Citations: 0

Abstract

The Na+/Ca2+ exchanger of Drosophila melanogaster, CALX, is the main Ca2+ -extrusion mechanism in olfactory sensory neurons and photoreceptor cells. Na+/Ca2+ exchangers have two Ca2+ sensor domains, CBD1 and CBD2. In contrast to the mammalian homologs, CALX is inhibited by Ca2+ binding to CALX-CBD1, whereas CALX-CBD2 does not bind Ca2+ at physiological concentrations. CALX-CBD1 consists of beta-sandwich and displays four Ca2+ -binding sites at the tip of the domain. In this study, we used NMR spectroscopy and isothermal titration calorimetry (ITC) to investigate the cooperativity of Ca2+ binding to CALX-CBD1. We observed that this domain binds Ca2+ in the slow exchange regime at the NMR chemical shift timescale. Ca2+ binding restricts the dynamics in the Ca2+-binding region. Experiments of N-15 chemical exchange saturation transfer and N-15 R(2 )dispersion allowed the determination of Ca2+ dissociation rates (similar to 30 s(-1)). NMR titration curves of residues in the Ca2+ binding region were sigmoidal because of the contribution of chemical exchange to transverse magnetization relaxation rates, R-2. Hence, a novel, to our knowledge, approach to analyze NMR titration curves was proposed. Ca2+-binding cooperativity was examined assuming two different stoichiometric binding models and using a Bayesian approach for data analysis. Fittings of NMR and ITC binding curves to the Hill model yielded n(Hill )similar to 2.9, near maximal cooperativity (n(Hill) = 4). By assuming a step-wise model to interpret the ITC data, we found that the probability of binding from 2 up to 4 Ca2+ is approximately three orders of magnitude higher than that of binding a single Ca2+. Hence, four Ca2+ ions bind almost simultaneously to CALX-CBD1. Cooperative Ca2+ binding is key to enable this exchanger to efficiently respond to changes in the intracellular Ca2+ concentration in sensory neuronal cells. (AU)

• FAPESP's process: 09/53989-4 - Acquisition of a nuclear magnetic resonance spectrometer for studies of biomolecules
• Grantee: Raghuvir Krishnaswamy Arni
• Support type: Multi-user Equipment Program
• FAPESP's process: 18/21450-8 - Cryo-EM Structural studies on the Xanthomonadaceae Type IV secretion systems
• Grantee: Jose David Rivera Echeverri
• Support type: Scholarships in Brazil - Post-Doctorate
• FAPESP's process: 17/05614-8 - Na +/Ca2+ exchangers: relationship between structure and function
• Grantee: Phelipe Augusto Mariano Vitale
• Support type: Scholarships in Brazil - Doctorate (Direct)
• FAPESP's process: 16/17375-5 - Solution nuclear magnetic resonance spectroscopy applied to study protein dynamics
• Grantee: Marcus Vinícius Cangussu Cardoso
• Support type: Scholarships in Brazil - Post-Doctorate
• FAPESP's process: 16/07490-1 - Structural studies of the Na+/Ca2+ exchanger and of proteins from the Zika virus by Nuclear Magnetic Resonance spectroscopy in solution
• Grantee: Roberto Kopke Salinas
• Support type: Regular Research Grants
• FAPESP's process: 17/06394-1 - Muti-user equipment approved in grant 2014/22136-4: isothermal titration calorimeter (ITC) for the determination of thermodynamic parameters of molecular interactions
• Grantee: Omar Abou El Seoud
• Support type: Multi-user Equipment Program