Ball-and-chain inactivation in a calcium-gated potassium channel

Inactivation is the process by which ion channels terminate ion flux through their pores while the opening stimulus is still present1. In neurons, inactivation of both sodium and potassium channels is crucial for the generation of action potentials and regulation of firing frequency1,2. A cytoplasmic domain of either the channel or an accessory subunit is thought to plug the open pore to inactivate the channel via a ‘ball-and-chain’ mechanism3–7. Here we use cryo-electron microscopy to identify the molecular gating mechanism in calcium-activated potassium channels by obtaining structures of the MthK channel from Methanobacterium thermoautotrophicum—a purely calcium-gated and inactivating channel—in a lipid environment. In the absence of Ca2+, we obtained a single structure in a closed state, which was shown by atomistic simulations to be highly flexible in lipid bilayers at ambient temperature, with large rocking motions of the gating ring and bending of pore-lining helices. In Ca2+-bound conditions, we obtained several structures, including multiple open-inactivated conformations, further indication of a highly dynamic protein. These different channel conformations are distinguished by rocking of the gating rings with respect to the transmembrane region, indicating symmetry breakage across the channel. Furthermore, in all conformations displaying open channel pores, the N terminus of one subunit of the channel tetramer sticks into the pore and plugs it, with free energy simulations showing that this is a strong interaction. Deletion of this N terminus leads to functionally non-inactivating channels and structures of open states without a pore plug, indicating that this previously unresolved N-terminal peptide is responsible for a ball-and-chain inactivation mechanism

Signal Transduction Mechanisms of HAMP and PAS Domains in Bacterial Chemotaxis

Bacteria utilize two-component systems to respond and adapt to changes in their environments. Central to the systems are modular receptors that comprise various functional domains to detect those changes and relay signals to effector domains. HAMP (Histidine kinases, Adenylate cyclases, Methyl accepting proteins and Phosphatases) and PAS (Per–Arnt–Sim) are two of the most common domains that couple various effectors to regulate a wide range of cellular activities. HAMP domains are signal relay modules that connects input and output domains. The HAMP domain from the E. coli serine receptor Tsr has been extensively studied by using genetic techniques, which leads to a model of HAMP biphasic stability that explains the behaviors of Tsr mutant receptors. However, limited biophysical data on the Tsr HAMP are available due to the instability of the domain. In order to provide stability to the Tsr HAMP, a chimera containing Tsr spliced into the poly-HAMP domains from Pseudomonas aeruginosa Aer2 (PaAer2) was created. Within the chimera, the Tsr HAMP maintains its characteristic four-helix coiled-coil structure with the distinctively lowered melting temperature compared to the PaAer2. This chimera was used to study three well-characterized HAMP mutational phenotypes differentiated by flagella-rotation patterns and CheA kinase activities: functional counterclockwise flagella rotation [CCW(A), kinase off], functional clockwise flagella rotation (CW, kinase on), and lesion-induced counterclockwise rotation [CCW(B), kinase off]. The stabilities and structural dynamics of the three phenotypes conform to the biphasic model. The transitions between functional on and off states are mediated by helix rotations and scissor-type movements. In the lesion-induced kinase off, the AS1 helices dissociate from the bundle while the AS2 helices form a two-helix colied coil. Overall, this study provides insights into relationships between HAMP conformational behaviors and their corresponding functional outputs. PAS domains are sensor motifs that are critical in signal transductions of prokaryotic and eukaryotic sensory proteins including chemoreceptors. Vibrio cholerae Aer2 (VcAer2), a PaAer2 homolog, has been shown to mediate responses to oxygen through the heme-binding PAS domains. Substitution of the conserved Trp 276 in the PAS2 domain to Leu abolished the O2-stabilizing ability, which corroborates its O2-ligating role. The crystal structure of the VcAer2 W276L is highly similar to the CN-bound PAS domain from PaAer2, suggesting the structure of the W276L mutant might represent the ligand-binding state. VcAer2 can serve as a promising alternative to E. coli Aer or PaAer2 for investigating PAS-mediated chemotaxis

H1D and H1 V33G receptors both respond to attractant, but with normal and inverse responses, respectively.

<p>(A) Swim assays of ATC receptors on tryptone agar plates. Colonies with functional chemoreceptors generate a characteristic ring near the leading edge of an expanding colony as cells consume Asp and swim towards higher Asp concentrations. H1 V33G generates an inverted ring, in comparison to Tar, which suggests an inverted CCW-to-CW response to Asp. (B) Temporal assays of transmembrane receptors showing response and adaptation kinetics. CheRB+ cells expressing various receptors were allowed to reach adaptation equilibrium before Asp was added. Tumbling frequencies alter if receptors are capable of receiving and relaying signal input from TM2 to the output KCM. Tar responds in the normal direction, switching from 12.5% to 1% CW bias. After ∼300 s, the adaptation system restores Tar CW bias to 12.5%. H1D has a normal Asp response, switching from 17.5% to 2.5% CW bias. H1 V33G displays an inverted response, switching from 16% to 100% CW bias upon Asp addition. A lower concentration of Asp is representative of increased receptor sensitivity.</p

HAMP Domain Conformers That Propagate Opposite Signals in Bacterial Chemoreceptors

<div><p>HAMP domains are signal relay modules in >26,000 receptors of bacteria, eukaryotes, and archaea that mediate processes involved in chemotaxis, pathogenesis, and biofilm formation. We identify two HAMP conformations distinguished by a four- to two-helix packing transition at the C-termini that send opposing signals in bacterial chemoreceptors. Crystal structures of signal-locked mutants establish the observed structure-to-function relationships. Pulsed dipolar electron spin resonance spectroscopy of spin-labeled soluble receptors active in cells verify that the crystallographically defined HAMP conformers are maintained in the receptors and influence the structure and activity of downstream domains accordingly. Mutation of HR2, a key residue for setting the HAMP conformation and generating an inhibitory signal, shifts HAMP structure and receptor output to an activating state. Another HR2 variant displays an inverted response with respect to ligand and demonstrates the fine energetic balance between “on” and “off” conformers. A DExG motif found in membrane proximal HAMP domains is shown to be critical for responses to extracellular ligand. Our findings directly correlate in vivo signaling with HAMP structure, stability, and dynamics to establish a comprehensive model for HAMP-mediated signal relay that consolidates existing views on how conformational signals propagate in receptors. Moreover, we have developed a rational means to manipulate HAMP structure and function that may prove useful in the engineering of bacterial taxis responses.</p> </div

Model for HAMP domain signal relay in bacterial chemoreceptors.

<p>The HAMP domains of MCPs exchange between HAMP1 and HAMP2 states to regulate bacterial chemotaxis. The conformation of HAMP2 imparts a two-helix coiled coil across the AS2/KCM junction, which results in CheA kinase inhibition and CCW flagella rotation. A dynamic HAMP1 forms a continuous four-helix coiled coil across the junction to generate kinase activation and CW flagella rotation.</p

Inter-spin distance measurements by PDS.

<p>Shown are experimentally determined distances of spin-labeled proteins and Cα–Cα distances from the Aer2 1–172 crystal structure. The values shown in parentheses refer to the width (Å) at half the maximum peak height, and qualify peak broadening and conformational heterogeneity. Small values represent narrow peaks and a homogeneous conformation. Large values represent broad peaks consistent with more heterogeneous populations.</p>a<p>Attachment of the MTSSL spin labels can add up to 13 Å to the Cα–Cα separation, or equivalently 6.5 Å each.</p

Signaling biases and expression levels of ATC receptors.

<p>(A) Structures of HAMP1 and HAMP2, highlighting positions of mutations reported in this study. HR2 (I88G) plays a prominent role in the HAMP2 hydrophobic core, inserting into the HAMP bundle between AS1 and AS2, while HR2 (V33G) in HAMP1 appears dispensable for bundle stability as it resides on the domain periphery. L21 and L44 occupy core heptad positions inside the HAMP bundle. Membrane-associated HAMP domains contain a highly conserved DExG motif at the connector-AS2 junction and a less conserved Pro residue between TM2 and AS1. (B) Tumbling biases of transmembrane and soluble ATC receptors quantified by temporal assays in CheRB+ and CheRB− cells. Signaling biases are grouped into four categories: (1) CCW locked (<5% CW), (2) slight CW bias (5%–10% CW), (3) CW bias (10%–50% CW), and (4) strong CW bias (50%–95% CW) or CW locked (>95% CW). Temporal assays confirm H1 and H1-2 induce opposite outputs. The L44H mutation generates a CW locked receptor with or without the adaptation system. The soluble receptors H1s and H1-2s generate more distinct CW and CCW locked phenotypes in CheRB− cells than their transmembrane counterparts. Mutation of HR2 in H1-2s I88G switches receptor signaling from CCW to CW locked, which is consistent with HR2 stabilizing the CCW HAMP2 conformer. (C) Expression levels of ATC receptors in CheRB+ (BT3388) cells, normalized to that of Tar for transmembrane receptors and that of Tar KCM for soluble receptors.</p