Crystal structure of dimeric cardiac L-type calcium channel regulatory domains bridged by Ca ²⁺ ·calmodulins

Abstract

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 ...

A framework for exhaustively mapping functional missense variants

“…Secondary structures were calculated using Stride (Frishman & Argos, 1995). Solvent accessibility and interface burial were calculated using the GETAREA tool (Fraczkiewicz & Braun, 1998) on the following PDB entries: for UBE2I: 3UIP (Gareau et al , 2012); 4W5V (Boucher et al unpublished); 3KYD (Olsen et al , 2010); 2UYZ (Knipscheer et al , 2007); 4Y1L (Alontaga et al , 2015); for SUMO1: 2G4D (Xu et al , 2006); 2IO2 (Reverter & Lima, 2006); 3KYD (Olsen et al , 2010); 3UIP (Gareau et al , 2012); 2ASQ (Song et al , 2005); 4WJO (Cappadocia et al , 2015); 4WJQ (Cappadocia et al , 2015); 1WYW (Baba et al , 2005); for calmodulin: 3G43 (Fallon  et al , 2009); 4DJC (Sarhan et al , 2012); and for TPK1: 3S4Y (Baker et al , 2001). …”

Structural Insights into Activation of the Retinal L-type Ca2+ Channel (Cav1.4) by Ca2+-binding Protein 4 (CaBP4)

“…The CaBP4 mutants M251A, F264E, and L268A showed at least 2-fold weaker binding with a significant loss in binding entropy, consistent with the removal of a hydrophobic interaction for each mutant ( Table 2). The CaBP4 C-lobe bound to the Cav1.4 IQ helix had the same relative orientation as the CaM C-lobe bound to the Cav1.2 IQ helix (49,50) (Fig. 8B).…”

“…Instead, an experimentally guided computational approach was used to dock the NMR structures of Ca 2ϩ -bound CaBP4 (Fig. 4) onto a modeled helical structure of the Cav1.4 IQ motif, derived from the helical Cav1.2 IQ motif seen in previous crystal structures (49,50). The helical structure of the IQ peptide bound to CaBP4 was confirmed by circular dichroism (Fig.…”

“…8A) 168 . The IQ helix bound to the CaBP4 N-lobe had an opposite orientation compared with the Cav1.2 IQ helix bound to the CaM N-lobe (49,50) (Fig. 8B).…”

Post-transcriptional modifications and “Calmodulation” of voltage-gated calcium channel function: Reflections by two collaborators of David T Yue

“…CaM fused protein naturally exists as a kinase in plant 38 and was successfully used for structural study by generating a CaM-MLCK derived peptide (M13) hybrid molecule, as reported by the Ikura's group. 39 Such hybrid proteins have a stoichiometry of 1:1 for CaM fused to the kinase or for CaM and target molecule such as MLCK. The original reason for the construction of the fusion channels was that they could be useful proteins for biochemical or structural studies such as to be used for protein crystallization.…”