Artigo Acesso aberto Revisado por pares

The second Ca 2+ -binding domain of the Na + –Ca 2+ exchanger is essential for regulation: Crystal structures and mutational analysis

2007; National Academy of Sciences; Volume: 104; Issue: 47 Linguagem: Inglês

10.1073/pnas.0707417104

ISSN

1091-6490

Autores

Gabriel Mercado Besserer, Michela Ottolia, Debora A. Nicoll, Vincent Chaptal, Duilio Cascio, Kenneth D. Philipson, Jeff Abramson,

Tópico(s)

Aluminum toxicity and tolerance in plants and animals

Resumo

The Na(+)-Ca(2+) exchanger plays a central role in cardiac contractility by maintaining Ca(2+) homeostasis. Two Ca(2+)-binding domains, CBD1 and CBD2, located in a large intracellular loop, regulate activity of the exchanger. Ca(2+) binding to these regulatory domains activates the transport of Ca(2+) across the plasma membrane. Previously, we solved the structure of CBD1, revealing four Ca(2+) ions arranged in a tight planar cluster. Here, we present structures of CBD2 in the Ca(2+)-bound (1.7-A resolution) and -free (1.4-A resolution) conformations. Like CBD1, CBD2 has a classical Ig fold but coordinates only two Ca(2+) ions in primary and secondary Ca(2+) sites. In the absence of Ca(2+), Lys(585) stabilizes the structure by coordinating two acidic residues (Asp(552) and Glu(648)), one from each of the Ca(2+)-binding sites, and prevents a substantial protein unfolding. We have mutated all of the acidic residues that coordinate the Ca(2+) ions and have examined the effects of these mutations on regulation of exchange activity. Three mutations (E516L, D578V, and E648L) at the primary Ca(2+) site completely remove Ca(2+) regulation, placing the exchanger into a constitutively active state. These are the first data defining the role of CBD2 as a regulatory domain in the Na(+)-Ca(2+) exchanger.

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