Ca2+-dependent inactivation (CDI) and facilitation (CDF) of the Ca(v)1.2 Ca2+ channel require calmodulin binding to a putative IQ motif in the carboxy-terminal tail of the pore-forming subunit. We present the 1.45 A crystal structure of Ca2+-calmodulin bound to a 21 residue peptide corresponding to the IQ domain of Ca(v)1.2. This structure shows that parallel binding of calmodulin to the IQ domain is governed by hydrophobic interactions. Mutations of residues I1672 and Q1673 in the peptide to alanines, which abolish CDI but not CDF in the channel, do not greatly alter the structure. Both lobes of Ca2+-saturated CaM bind to the IQ peptide but isoleucine 1672, thought to form an intramolecular interaction that drives CDI, is buried. These findings suggest that this structure could represent the conformation that calmodulin assumes in CDF.
Calmodulin has been a subject of intense scrutiny since its discovery because of its unusual properties in regulating the functions of about 100 diverse target enzymes and structural proteins. The original and to date only crystal conformation of native eukaryotic Ca(2+)-calmodulin (Ca(2+)-CaM) is a very extended molecule with two widely separated globular domains linked by an exposed long helix. Here we report the 1.7 A X-ray structure of a new native Ca(2+)-CaM that is in a compact ellipsoidal conformation and shows a sharp bend in the linker helix and a more contracted N-terminal domain. This conformation may offer advantages for recognition of kinase-type calmodulin targets or small organic molecule drugs.
Voltage-dependent calcium channels (CaV) open in response to changes in membrane potential, but their activity is modulated by Ca 2؉ binding to calmodulin (CaM). Structural studies of this family of channels have focused on CaM bound to the IQ motif; however, the minimal differences between structures cannot adequately describe CaM's role in the regulation of these channels. We report a unique crystal structure of a 77-residue fragment of the Ca V1.2 ␣1 subunit carboxyl terminus, which includes a tandem of the pre-IQ and IQ domains, in complex with Ca 2؉ ⅐CaM in 2 distinct binding modes. The structure of the Ca V1.2 fragment is an unusual dimer of 2 coiled-coiled pre-IQ regions bridged by 2 Ca 2؉ ⅐CaMs interacting with the pre-IQ regions and a canonical Ca V1-IQ-Ca 2؉ ⅐CaM complex. Native Ca V1.2 channels are shown to be a mixture of monomers/dimers and a point mutation in the pre-IQ region predicted to abolish the coiled-coil structure significantly reduces Ca 2؉ -dependent inactivation of heterologously expressed CaV1.2 channels.structure ͉ function ͉ voltage-gated calcium channel E xcitation-contraction coupling and other important cellular processes are controlled by the voltage-gated Ca 2ϩ channels (Ca V ). The ubiquitous Ca 2ϩ sensor and regulator molecule, calmodulin, is an essential component of Ca V regulation by Ca 2ϩ , and several regions of the cytoplasmic carboxyl terminus of the Ca V ␣1 subunit have been identified as critical molecular determinants for CaM's regulation of Ca V . Ca 2ϩ ⅐CaM bound to the IQ motif of the carboxyl terminus of the ␣ 1 subunit of L-type Ca 2ϩ channels is required for both a feed-forward regulation, Ca 2ϩ -dependent facilitation (CDF), and a feed-back regulation, Ca 2ϩ -dependent inactivation (CDI) (1, 2). CaM acts as the Ca 2ϩ sensor for CDI in Ca V 1.2, Ca V 2.1, Ca V 2.2 and Ca V 2.3, and CDF in Ca V 1.2 and Ca V 2.1. This duality of Ca V regulation by CaM suggests that there are either multiple binding sites or alternative interactions exist to regulate the channel based on different functional states of the channel.Regions upstream of the IQ motif, designated the pre-IQ region, have been implicated in Ca 2ϩ ⅐CaM regulation of the channel (3-5). Consistent with this, 2 different segments within the pre-IQ motif (1606-1627 and 1618-1652, identified as the A and C sequences, respectively) have been shown to bind CaM (4). Moreover, both the IQ motif and amino acids within the pre-IQ region (N1630-E1631) have been indicated to be critical for Ca V 1.2 CDI (6).Recent crystal structures of CaM in complex with the IQ motifs from Ca V 1.2 (7, 8), Ca V 2.2 and Ca V 2.3 (5) reveal few structural differences that could account for the differences in regulation of Ca V 1.2 and Ca V 2.2 by CaM. However, very little is known about the structure of the pre-IQ region or the molecular basis for its interactions with CaM.Here, we report the isolation and determination of the crystal structure at 2.1 Å resolution of a 77-residue (1609-1685) fragment of the carboxyl terminus of the ␣ 1 ...
Calmodulin binds to IQ motifs in the
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