Characterization of the C-terminal Domain of a Potassium Channel from Streptomyces lividans (KcsA)

Pau, Victor P.T.; Zhu, Yongfang; Yuchi, Zhiguang; Hoang, Quyen Q.; Yang, Dan

doi:10.1074/jbc.m703277200

Cited by 19 publications

(41 citation statements)

References 24 publications

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“…H4 in WSK3 is indeed protonated at pH Ϸ4.5 used in our studies, as confirmed by the chemical shifts of the imidazole H␦2 and H 1 (7.22 and 8.53 ppm, respectively). Similarly, the carboxy-terminal segment, when present, also modulates pore opening in response to decreasing pH, apparently by weakening the stability of the closed state at low pH (23). Thus, the cytoplasmic vestibule of the WSK3 pore, lacking the stability provided by a membrane environment, assumes a configuration that perhaps bears more resemblance to the state of transition to an open channel.…”

Section: Discussionmentioning

confidence: 99%

NMR studies of a channel protein without membranes: Structure and dynamics of water-solubilized KcsA

Ma¹,

Tillman²,

Tang³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Structural studies of polytopic membrane proteins are often hampered by the vagaries of these proteins in membrane mimetic environments and by the difficulties in handling them with conventional techniques. Designing and creating water-soluble analogues with preserved native structures offer an attractive alternative. We report here solution NMR studies of WSK3, a water-soluble analogue of the potassium channel KcsA. The WSK3 NMR structure (PDB ID code 2K1E) resembles the KcsA crystal structures, validating the approach. By more stringent comparison criteria, however, the introduction of several charged residues aimed at improving water solubility seems to have led to the possible formations of a few salt bridges and hydrogen bonds not present in the native structure, resulting in slight differences in the structure of WSK3 relative to KcsA. NMR dynamics measurements show that WSK3 is highly flexible in the absence of a lipid environment. Reduced spectral density mapping and model-free analyses reveal dynamic characteristics consistent with an isotropically tumbling tetramer experiencing slow (nanosecond) motions with unusually low local ordering. An altered hydrogen-bond network near the selectivity filter and the pore helix, and the intrinsically dynamic nature of the selectivity filter, support the notion that this region is crucial for slow inactivation. Our results have implications not only for the design of water-soluble analogues of membrane proteins but also for our understanding of the basic determinants of intrinsic protein structure and dynamics. membrane protein ͉ protein design ͉ potassium channels ͉ slow inactivation L ess than 1% of known protein structures belong to transmembrane (TM) proteins. The scarcity of structural data is due to the difficulties associated with handling membrane proteins for structure determination. With few exceptions, membrane proteins are often present at low levels in natural tissues, and the available cellular machinery for membrane insertion often limits the capacity of high-level expression systems. Outside their native environment, membrane proteins are unpredictable in behavior, with proper folding and stability depending on the choice of the membrane mimetic environment in addition to the common variables affecting soluble proteins. Although some membrane proteins can be correctly refolded after misfolding or aggregation during expression and purification, others are affected by these processes irreversibly. Currently, no reliable method is available to predict which membrane mimetic will stabilize a given protein in its native conformation without aggregation, and often an arduous process of trial and error using expensive reagents must be carried out to find appropriate conditions.An intriguing alternative to inserting TM proteins into a membrane mimetic environment for structure determination is to alter the proteins such that a membrane is no longer required. Designing water-soluble analogues of membrane proteins challenges the basic understanding of what st...

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Section: Discussionmentioning

confidence: 99%

NMR studies of a channel protein without membranes: Structure and dynamics of water-solubilized KcsA

Ma¹,

Tillman²,

Tang³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…The rapid cytosolic acidification that should follow CCCP addition might similarly affect ScMep2 activity. Nor can we exclude that pH variations might modulate a gating mechanism involving the C termini of Mep proteins, as shown for the potassium channel KcsA of Streptomyces lividans (51).…”

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confidence: 99%

Distinct Transport Mechanisms in Yeast Ammonium Transport/Sensor Proteins of the Mep/Amt/Rh Family and Impact on Filamentation

Boeckstaens¹,

André

Marini

2008

Journal of Biological Chemistry

113

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Ammonium transport proteins of the Mep/Amt/Rh family include microbial and plant Mep/Amt members, crucial for ammonium scavenging, and animal Rhesus factors likely involved in ammonium disposal. Recent structural information on two bacterial Mep/Amt proteins has revealed the presence, in the hydrophobic conducting pore, of a pair of preserved histidines proposed to play an important role in substrate conductance, by participating either in NH 4 ؉ deprotonation or in shaping the pore. Here we highlight the existence of two functional Mep/Amt subfamilies distinguishable according to whether the first of these histidines is conserved, as in yeast ScMep2, or replaced by glutamate, as in ScMep1. Replacement of the native histidine of ScMep2 with glutamate leads to conversion from ScMep2 to ScMep1-like properties. This includes a two-unit upshift of the optimal pH for transport and an increase of the transport rate, consistent with alleviation of an energy-limiting step. Similar effects are observed when the same substitution is introduced into the Escherichia coli AmtB protein. In contrast to ScMep1, ScMep2 is proposed to play an additional signaling role in the induction of filamentous growth, a dimorphic change often associated with virulence in pathogenic fungi. We show here that the histidine to glutamate substitution in ScMep2 leads to uncoupling of the transport and sensor functions, suggesting that a ScMep2-specific transport mechanism might be responsible for filamentation. Our overall data suggest the existence of two functional groups of Mep/Amt-type proteins with different transport mechanisms and distinct impacts on cell physiology and signaling.

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“…EPR spectroscopy | K+ channel | X-ray crystallography M ost ion channels have structured cytoplasmic domains that influence their functional behavior [in regards to both gating (1-3) and permeation (4)], contribute to their structural stability (5,6) or allow them to directly interact with enzymes and other regulators (7). In the prokaryotic K þ channel KcsA, the 40-residue C-terminus forms a four-helix bundle that projects toward the cytoplasm (5).…”

mentioning

confidence: 99%

“…In the prokaryotic K þ channel KcsA, the 40-residue C-terminus forms a four-helix bundle that projects toward the cytoplasm (5). Removal of the C-terminus affects KcsA thermal stability, destabilizes the closed conformation (5,6) and enhances entry into the C-type inactivated state (8). Using chaperone-assisted crystallography, we recently determined the crystal structure of full-length (FL) KcsA in its closed conformation (1).…”

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confidence: 99%

Mechanism of activation gating in the full-length KcsA K ⁺ channel

Uysal

Cuello

Cortés

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Using a constitutively active channel mutant, we solved the structure of full-length KcsA in the open conformation at 3.9 Å. The structure reveals that the activation gate expands about 20 Å, exerting a strain on the bulge helices in the C-terminal domain and generating side windows large enough to accommodate hydrated K þ ions. Functional and spectroscopic analysis of the gating transition provides direct insight into the allosteric coupling between the activation gate and the selectivity filter. We show that the movement of the inner gate helix is transmitted to the C-terminus as a straightforward expansion, leading to an upward movement and the insertion of the top third of the bulge helix into the membrane. We suggest that by limiting the extent to which the inner gate can open, the cytoplasmic domain also modulates the level of inactivation occurring at the selectivity filter.EPR spectroscopy | K+ channel | X-ray crystallography M ost ion channels have structured cytoplasmic domains that influence their functional behavior [in regards to both gating (1-3) and permeation (4)], contribute to their structural stability (5, 6) or allow them to directly interact with enzymes and other regulators (7). In the prokaryotic K þ channel KcsA, the 40-residue C-terminus forms a four-helix bundle that projects toward the cytoplasm (5). Removal of the C-terminus affects KcsA thermal stability, destabilizes the closed conformation (5, 6) and enhances entry into the C-type inactivated state (8). Using chaperone-assisted crystallography, we recently determined the crystal structure of full-length (FL) KcsA in its closed conformation (1). The FL KcsA structure revealed that the C-terminal domain forms a mixed twofold/fourfold symmetric, four-helix bundle that projects approximately 70 Å toward the cytoplasm and applies a steric bias on the gate, tightening the inner bundle gate and stabilizing the closed conformation (1).In KcsA, proton dependent gating is accompanied by large movements in the helical transmembrane segment TM2. These rearrangements have been fully characterized by spectroscopic methods [EPR (9), fluorescence (10), NMR (11)], mass tagging (12), surface plasmon resonance(13) and X-ray/neutron scattering (14), and are fully consistent with available open K þ channel crystallographic structures (15,16). However, the extent of the inner bundle gate opening, the basic set of C-terminus activation gating conformational changes and the physiological ion pathway during permeation are still not well understood in the full-length channel. KcsA (15), and second, of the availability of antibody fragments directed toward the C-terminal bundle (1). Crystals of FL KcsA-Fab2 were obtained in the orthorhombic space group I222 with two Fabs bound per tetramer and diffracted to 3.9 Å. Importantly, these crystals were highly isomorphous with the FL KcsA-Fab2 closed form (3EFF) reported earlier. Thus, a direct comparison between the open and closed forms could be made in the same unit cell using Fourier difference maps. This allo...

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Characterization of the C-terminal Domain of a Potassium Channel from Streptomyces lividans (KcsA)

Cited by 19 publications

References 24 publications

NMR studies of a channel protein without membranes: Structure and dynamics of water-solubilized KcsA

NMR studies of a channel protein without membranes: Structure and dynamics of water-solubilized KcsA

Distinct Transport Mechanisms in Yeast Ammonium Transport/Sensor Proteins of the Mep/Amt/Rh Family and Impact on Filamentation

Mechanism of activation gating in the full-length KcsA K ⁺ channel

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Characterization of the C-terminal Domain of a Potassium Channel from Streptomyces lividans (KcsA)

Cited by 19 publications

References 24 publications

NMR studies of a channel protein without membranes: Structure and dynamics of water-solubilized KcsA

NMR studies of a channel protein without membranes: Structure and dynamics of water-solubilized KcsA

Distinct Transport Mechanisms in Yeast Ammonium Transport/Sensor Proteins of the Mep/Amt/Rh Family and Impact on Filamentation

Mechanism of activation gating in the full-length KcsA K + channel

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Mechanism of activation gating in the full-length KcsA K ⁺ channel