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 COOHterminal 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 firstcoiled-coilcomplexisdimericandundergoesconcentrationdependent 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 Å crystallographic data. Both coiledcoils 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.

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 Å 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.

Voltage-dependent calcium channels selectively enable Ca 2؉ ion movement through cellular membranes. These multiprotein complexes are involved in a wide spectrum of biological processes such as signal transduction and cellular homeostasis. ␣ 1 is the membrane pore-forming subunit, whereas ␤ is an intracellular subunit that binds to ␣ 1 , facilitating and modulating channel function. We have expressed, purified, and characterized recombinant ␤ 3 and ␤ 2a using both biochemical and biophysical methods, including electrophysiology, to better understand the ␤ family's protein structural and functional correlates. Our results indicate that the ␤ 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 ␤ is not in complex with the channel. In addition, the ␤ structural core, comprised of just these two domains without other sequences, binds tightly to the ␣ interaction domain (AID) motif, a sequence derived from the ␣ 1 subunit and the principal anchor site of ␤. Domain II is responsible for this binding, but domain I enhances it.Voltage-dependent calcium channels (VDCCs) 1 permit the flow of Ca 2ϩ ions through cellular membranes as a function of membrane potential. These protein complexes are central components in a variety of physiological systems of organisms, ranging from yeast to human. They play pivotal roles in signal transduction and homeostasis processes.Functional roles for these channels vary based on cell type. In muscle, both skeletal and cardiac, the predominant VDCCs (Ca V 1.1 and Ca V 1.2) cause release of Ca 2ϩ into the cytosol from intracellular stores, thereby initiating contraction (1, 2). In neurons and endocrine cells, neurotransmitter or hormone secretion requires VDCC activity. In addition, electrical activity, specifically the action potential in cardiac myocytes, is regulated by VDCCs. Finally, calcium influx and concentration controlled by VDCCs plays a significant role in neuronal gene expression (3). Pathways have been elucidated, where for one example, VDCC activity gives rise to phosphorylation of CREB, a transcription factor (4), thereby activating transcription of a myriad of target genes.The VDCC comprises four distinct polypeptides: ␣ 1 , ␣ 2 ␦, ␤, and ␥ (5). ␣ 1 is the membrane pore-forming subunit and numbers between 1800 and 2400 residues in length. Its sequence exhibits repeats comprising four transmembrane modules or domains, akin to the tetrameric architecture of potassium channels. Each module contains the canonical transmembrane arrangement for voltage-gated ion channels i.e. six transmembrane segments. Modules are connected by linkers that are located in the intracellular milieu, as are both the N and C termini. The high voltage-activated channel subunits, Ca V 1.x and Ca V 2.x (␣ 1 ), numbering seven in total, share a high degree of sequence similarity but nevertheless encode distinct electrophysiological activities.The ␤ subunit was firs...

3,4 ✉ 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:BeF 3 -bound) ground-state mimic double-ring mHsp60 14 -(mHsp10 7 ) 2 football complex, and the cryo-EM structures of the ADP-bound successor mHsp60 14 -(mHsp10 7 ) 2 complex, and a single-ring mHsp60 7 -mHsp10 7 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 halffootballs. 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.

Voltage-dependent calcium channels (VDCCs) allow the passage of Ca 2ϩ ions through cellular membranes in response to membrane depolarization. The channel pore-forming subunit, ␣1, and a regulatory subunit (Ca V ␤) form a high affinity complex where Ca V ␤ binds to a ␣1 interacting domain in the intracellular linker between ␣1 membrane domains I and II (I-II linker). We determined crystal structures of Ca V ␤2 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 ␤ 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 ␤, 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.