Reconstitution of ß-adrenergic regulation of CaV1.2: Rad-dependent and Rad-independent protein kinase A mechanisms.

L-type voltage-gated CaV1.2 channels crucially regulate cardiac muscle contraction. Activation of ß-adrenergic receptors (ß-AR) augments contraction via protein kinase A (PKA)-induced increase of calcium influx through CaV1.2 channels. To date, the full ß-AR cascade has never been heterologously reconstituted. A recent study identified Rad, a CaV1.2 inhibitory protein, as essential for PKA regulation of CaV1.2. We corroborated this finding and reconstituted the complete pathway with agonist activation of ß1-AR or ß2-AR in Xenopus oocytes. We found, and distinguished between, two distinct pathways of PKA modulation of CaV1.2: Rad dependent (∼80% of total) and Rad independent. The reconstituted system reproduces the known features of ß-AR regulation in cardiomyocytes and reveals several aspects: the differential regulation of posttranslationally modified CaV1.2 variants and the distinct features of ß1-AR versus ß2-AR activity. This system allows for the addressing of central unresolved issues in the ß-AR-CaV1.2 cascade and will facilitate the development of therapies for catecholamine-induced cardiac pathologies.

Structural basis for active single and double ring complexes in human mitochondrial Hsp60-Hsp10 chaperonin.

mHsp60-mHsp10 assists the folding of mitochondrial matrix proteins without the negative ATP binding inter-ring cooperativity of GroEL-GroES. Here we report the crystal structure of an ATP (ADP:BeF3-bound) ground-state mimic double-ring mHsp6014-(mHsp107)2 football complex, and the cryo-EM structures of the ADP-bound successor mHsp6014-(mHsp107)2 complex, and a single-ring mHsp607-mHsp107 half-football. The structures explain the nucleotide dependence of mHsp60 ring formation, and reveal an inter-ring nucleotide symmetry consistent with the absence of negative cooperativity. In the ground-state a two-fold symmetric H-bond and a salt bridge stitch the double-rings together, whereas only the H-bond remains as the equatorial gap increases in an ADP football poised to split into half-footballs. Refolding assays demonstrate obligate single- and double-ring mHsp60 variants are active, and complementation analysis in bacteria shows the single-ring variant is as efficient as wild-type mHsp60. Our work provides a structural basis for active single- and double-ring complexes coexisting in the mHsp60-mHsp10 chaperonin reaction cycle.

Ca(V)1.2 I-II linker structure and Timothy syndrome.

Ca(V) channels are multi-subunit protein complexes that enable inward cellular Ca(2+) currents in response to membrane depolarization. We recently described structure-function studies of the intracellular α1 subunit domain I-II linker, directly downstream of domain IS6. The results show the extent of the linker's helical structure to be subfamily dependent, as dictated by highly conserved primary sequence differences. Moreover, the difference in structure confers different biophysical properties, particularly the extent and kinetics of voltage and calcium-dependent inactivation. Timothy syndrome is a human genetic disorder due to mutations in the Ca(V)1.2 gene. Here, we explored whether perturbation of the I-II linker helical structure might provide a mechanistic explanation for a Timothy syndrome mutant's (human Ca(V)1.2 G406R equivalent) biophysical effects on inactivation and activation. The results are equivocal, suggesting that a full mechanistic explanation for this Timothy syndrome mutation requires further investigation.

The role of a voltage-dependent Ca2+ channel intracellular linker: a structure-function analysis.

Voltage-dependent calcium channels (VDCCs) allow the passage of Ca(2+) ions through cellular membranes in response to membrane depolarization. The channel pore-forming subunit, α1, and a regulatory subunit (Ca(V)ß) form a high affinity complex where Ca(V)ß binds to a α1 interacting domain in the intracellular linker between α1 membrane domains I and II (I-II linker). We determined crystal structures of Ca(V)ß2 functional core in complex with the Ca(V)1.2 and Ca(V)2.2 I-II linkers to a resolution of 1.95 and 2.0 Å, respectively. Structural differences between the highly conserved linkers, important for coupling Ca(V)ß to the channel pore, guided mechanistic functional studies. Electrophysiological measurements point to the importance of differing linker structure in both Ca(V)1 and 2 subtypes with mutations affecting both voltage- and calcium-dependent inactivation and voltage dependence of activation. These linker effects persist in the absence of Ca(V)ß, pointing to the intrinsic role of the linker in VDCC function and suggesting that I-II linker structure can serve as a brake during inactivation.

The Arabidopsis COP9 signalosome subunit 7 is a model PCI domain protein with subdomains involved in COP9 signalosome assembly.

The COP9 Signalosome (CSN) is a multiprotein complex that was originally identified in Arabidopsis thaliana as a negative regulator of photomorphogenesis and subsequently shown to be a general eukaryotic regulator of developmental signaling. The CSN plays various roles, but it has been most often implicated in regulating protein degradation pathways. Six of eight CSN subunits bear a sequence motif called PCI. Here, we report studies of subunit 7 (CSN7) from Arabidopsis, which contains such a motif. Our in vitro and structural results, based on 1.5 A crystallographic data, enable a definition of a PCI domain, built from helical bundle and winged helix subdomains. Using functional binding assays, we demonstrate that the PCI domain (residues 1 to 169) interacts with two other PCI proteins, CSN8 and CSN1. CSN7 interactions with CSN8 use both PCI subdomains. Furthermore, we show that a C-terminal tail outside of this PCI domain is responsible for association with the non-PCI subunit, CSN6. In vivo studies of transgenic plants revealed that the overexpressed CSN7 PCI domain does not assemble into the CSN, nor can it complement a null mutation of CSN7. However, a CSN7 clone that contains the PCI domain plus part of the CSN6 binding domain can complement the null mutation in terms of seedling viability and photomorphogenesis. These transgenic plants, though, are defective in adult growth, suggesting that the CSN7 C-terminal tail plays additional functional roles. Together, the findings have implications for CSN assembly and function, highlighting necessary interactions between subunits.

The KCNQ1 (Kv7.1) COOH terminus, a multitiered scaffold for subunit assembly and protein interaction.

The Kv7 subfamily of voltage-dependent potassium channels, distinct from other subfamilies by dint of its large intracellular COOH terminus, acts to regulate excitability in cardiac and neuronal tissues. KCNQ1 (Kv7.1), the founding subfamily member, encodes a channel subunit directly implicated in genetic disorders, such as the long QT syndrome, a cardiac pathology responsible for arrhythmias. We have used a recombinant protein preparation of the COOH terminus to probe the structure and function of this domain and its individual modules. The COOH-terminal proximal half associates with one calmodulin constitutively bound to each subunit where calmodulin is critical for proper folding of the whole intracellular domain. The distal half directs tetramerization, employing tandem coiled-coils. The first coiled-coil complex is dimeric and undergoes concentration-dependent self-association to form a dimer of dimers. The outer coiled-coil is parallel tetrameric, the details of which have been elucidated based on 2.0 A crystallographic data. Both coiled-coils act in a coordinate fashion to mediate the formation and stabilization of the tetrameric distal half. Functional studies, including characterization of structure-based and long QT mutants, prove the requirement for both modules and point to complex roles for these modules, including folding, assembly, trafficking, and regulation.

Structure-function studies of the G-domain from human gem, a novel small G-protein.

Gem, a member of the Rad,Gem/Kir subfamily of small G-proteins, has unique sequence features. We report here the crystallographic structure determination of the Gem G-domain in complex with nucleotide to 2.4 A resolution. Although the basic Ras protein fold is maintained, the Gem switch regions emphatically differ from the Ras paradigm. Our ensuing biochemical characterization indicates that Gem G-domain markedly prefers GDP over GTP. Two known functions of Gem are distinctly affected by spatially separated clusters of mutations.

Expression, purification and crystallization of a functional core of the voltage-dependent calcium channel beta subunit.

Two versions of the functional core of the rabbit voltage-dependent calcium channel beta2a subunit were expressed in Escherichia coli. These proteins were purified to homogeneity and screened for crystallization. Crystallization conditions were refined using the hanging-drop vapour-diffusion method and two crystal forms were pursued. Crystal form I is represented by thick rods with tetragonal symmetry, unit-cell parameters a = b = 75, c = 165 A and a diffraction limit of 3.4 A which were obtained using ammonium sulfate as a precipitant. Crystal form II gives rise to plates with orthorhombic symmetry, unit-cell parameters a = 35, b = 75, c = 165 A and a diffraction limit of 2.3 A which were grown using polyethylene glycol 20K as a precipitant.

The voltage-dependent calcium channel beta subunit contains two stable interacting domains.

Voltage-dependent calcium channels selectively enable Ca2+ ion movement through cellular membranes. These multiprotein complexes are involved in a wide spectrum of biological processes such as signal transduction and cellular homeostasis. alpha1 is the membrane pore-forming subunit, whereas beta is an intracellular subunit that binds to alpha1, facilitating and modulating channel function. We have expressed, purified, and characterized recombinant beta3 and beta2a using both biochemical and biophysical methods, including electrophysiology, to better understand the beta family's protein structural and functional correlates. Our results indicate that the beta protein is composed of two distinct domains that associate with one another in a stable manner. The data also suggest that the polypeptide regions outside these domains are not structured when beta is not in complex with the channel. In addition, the beta structural core, comprised of just these two domains without other sequences, binds tightly to the alpha interaction domain (AID) motif, a sequence derived from the alpha1 subunit and the principal anchor site of beta. Domain II is responsible for this binding, but domain I enhances it.