Structure and Function of Membrane Proteins

National Institute Of Neurological Disorders And Stroke

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Abstract

Calcium ions increase the open probability of the large conductance potassium (BK) channels by binding to high affinity sites within the large cytosolic Ca2+ sensing structure called the gating ring. This Ca2+ sensing structure is formed by eight Regulator of K+ Conductance (RCK) domains, two from each subunit of the channel, termed them RCK1 and RCK2. Other divalent species, like Ba2+ ions, are also able to activate BK channels by interacting with the Ca2+ sensor. Unlike Ca2+ that activate BK channel through all RCK domains, Ba2+ does it selectively through the RCK2 domain. In addition to activation, Ba2+ can also block K+ permeation by binding to the selectivity filter of BK channels. We have solved Cryo-EM structures of BK channels with Ba2+. Structurally, blockade arises from one Ba2+ occupying the traditional K+ site S3 in the selectivity filter of the channel. In addition, electronic densities attributed to K+ ions are detected at K+ sites S2 and S4. In the gating ring, eight Ba2+ ions are bound to the corresponding high affinity Ca2+ binding sites. Thereby, the lack of functional significance of the RCK1 on the activation by Ba2+ should arise because the architectures of the gating rings in our structures represent intermediate transitions between the close and the open configurations. Finally, these Ba2+ structures reveal an intricate series of concerted changes of the RCK 1 and RCK2 from the same subunit, suggesting coordinated intra-subunit dynamics. The Na+/K+-ATPase is a membrane embedded enzyme responsible for the exchange of 3 intracellular Na+ for 2 extracellular K+, fueled by the hydrolysis of 1 molecule of ATP. By performing this transport process, the Na+/K+-ATPase maintains the electrochemical gradients of these ions between the internal and external environments of animal cells, which are used for action potential signaling and for cotransport of substrates such as calcium, nutrients, and neurotransmitters. Reduction of Na+/K+-ATPase activity thus causes disease symptoms attributable to impairment of action potentials, disruption of other cellular functions, and cell death. Pathogenic variants in the ATP1A3 Na+/K+-ATPase gene, which encodes an isoform of the catalytic subunit that is highly expressed in neural and heart tissues, cause disabling neurological diseases with varying cardiac involvement. When ATP1A3 residues that participate in ion binding are involved, the resulting phenotypes often fall within the spectrum of rapid-onset dystonia-parkinsonism (RDP) to alternating hemiplegia of childhood (AHC). AHC and RDP are broadly characterized by asymmetric movement disorders plus other features such as plegia, hypotonia, intellectual disability, epilepsy, and cardiac arrhythmia in patients toward the more severe AHC end of the spectrum. The core enzyme is an obligatory heterodimer of an and subunit, in which the subunit contains the ATP binding domain as well as all three ion binding sites. Normal export of Na+ through the Albers-Post enzymatic cycle starts with the binding of 3 intracellular Na+ to the ATP-bound E1 form of the enzyme, which has high Na+ affinity (Na3-E1-ATP). These Na+ occupy 3 sites, numbered I-III in the crystal structure. Binding of all 3 Na+ triggers occlusion, enzyme phosphorylation with release of ADP, and change to a conformation that has an extracellular ion pathway and high affinity for K+ (P-E2-Na3). Extracellular Na+ release occurs sequentially with deocclusion followed by stepwise unbinding of each of the 3 Na+ at different rates. After releasing Na+, the phosphorylated E2 enzyme binds 2 K+ at sites I and II and imports them in a series of steps involving dephosphorylation and ATP binding. We studied mutations of three ATP1A3 residues thought to exclusively coordinate Na+ but not K+: Y768, T771, and D923. Missense variants of the first two (Y768C, Y768H, T771I, T771N) cause the severe phenotype, AHC, but their functional effects have not previously been characterized in ATP1A3. Missense variants of D923 (D923N, D923Y) cause phenotypes ranging across the AHC/RDP spectrum and reduce ion transport, with a reduction in the overall apparent affinity for Na+ observed for D923N. From Na+-bound crystal structures, the side chains of D923, Y768 and T771 specifically participate in the coordination of one Na+ at site III. Unlike sites I and II which are shared by K+ during the transport cycle, site III is uniquely used by one Na+. The ongoing COVID-19 pandemic caused by sever acute respiratory syndrome coronavirus 2 (SARS-CoV-2) constituted a serious threat to global human health and underscored the need of detailed research into the SARS-CoV-2 virus. This virus which had high sequence similarity to SARS-CoV and bat-borne coronaviruses encodes four structural proteins, sixteen non-structural proteins and nine accessory proteins for viral replication and release. Structural proteins, including spike (S) protein, membrane (M) protein, envelope (E) protein, and nucleocapsid (N) protein, are incorporated into the structurally complete viral particles. Non-structural proteins act as various enzymes and transcription factors for viral replication and pathogenicity. Accessory proteins perform a diverse range of functions, involved in viral release, stability, and pathogenesis. Among these accessory proteins in SARS-CoV-2, ORF3a which is located between S and E proteins on the 3 terminal genome is the largest. It is also a membrane protein, present exclusively in SARS-like coronavirus but no other viruses. SARS-CoV-2 ORF3a had a high sequence identity (73%) with its SARS-CoV homolog, particularly in functional domains, such as the 3 transmembrane (TM) segments, suggesting functional similarity among the homologous ORF3a proteins. We are focusing our study to four aspects of the biology of ORF3a: 1) its oligoramization state in mammalian cells, 2) its functional properties as an ion channel, 3) its localization within the infected cells, and 4) its proteomic vicinity.