Homology modeling and molecular dynamics simulation studies of an inward rectifier potassium channel

A homology model has been generated for the pore-forming domain of Kir6.2, a component of an ATP-sensitive K channel, based on the x-ray structure of the bacterial channel KcsA. Analysis of the lipid-exposed and pore-lining surfaces of the model reveals them to be compatible with the known features of membrane proteins and Kir channels, respectively. The Kir6.2 homology model was used as the starting point for nanosecond-duration molecular dynamics simulations in a solvated phospholipid bilayer. The overall drift from the model structure was comparable to that seen for KcsA in previous similar simulations. Preliminary analysis of the interactions of the Kir6.2 channel model with K(+) ions and water molecules during these simulations suggests that concerted single-file motion of K(+) ions and water through the selectivity filter occurs. This is similar to such motion observed in simulations of KcsA. This suggests that a single-filing mechanism is conserved between different K channel structures and may be robust to changes in simulation details. Comparison of Kir6.2 and KcsA suggests some degree of flexibility in the filter, thus complicating models of ion selectivity based upon a rigid filter.     

Potassium channel, ions, and water: simulation studies based on the high resolution X-ray structure of KcsA

Interactions of Na(+), K(+), Rb(+), and Cs(+) ions within the selectivity filter of a potassium channel have been investigated via multiple molecular dynamics simulations (total simulation time, 48 ns) based on the high resolution structure of KcsA, embedded in a phospholipid bilayer. As in simulations based on a lower resolution structure of KcsA, concerted motions of ions and water within the filter are seen. Despite the use of a higher resolution structure and the inclusion of four buried water molecules thought to stabilize the filter, this region exhibits a significant degree of flexibility. In particular, pronounced distortion of filter occurs if no ions are present within it. The two most readily permeant ions, K(+) and Rb(+), are similar in their interactions with the selectivity filter. In contrast, Na(+) ions tend to distort the filter by binding to a ring of four carbonyl oxygens. The larger Cs(+) ions result in a small degree of expansion of the filter relative to the x-ray structure. Cs(+) ions also appear to interact differently with the gate region of the channel, showing some tendency to bind within a predominantly hydrophobic pocket. The four water molecules buried between the back of the selectivity filter and the remainder of the protein show comparable mobility to the surrounding protein and do not exchange with water molecules within the filter or the central cavity. A preliminary comparison of the use of particle mesh Ewald versus cutoff protocols for the treatment of long-range electrostatics suggests some difference in the kinetics of ion translocation within the filter.     

Dynamics, Energetics, and Selectivity of the Low-K KcsA Channel Structure

Potassium channels are a diverse family of integral membrane proteins through which K⁺ can pass selectively. There is ongoing debate about the nature of conformational changes associated with the opening/closing and conductive/nonconductive states of potassium channels. The channels partly exert their function by varying their conductance through a mechanism known as C-type inactivation. Shortly after the activation of K⁺ channels, their selectivity filter stops conducting ions at a rate that depends on various stimuli. The molecular mechanism of C-type inactivation has not been fully understood yet. However, the X-ray structure of the KcsA channel obtained in the presence of low K⁺ concentration is thought to be representative of a K⁺ channel in the C-type inactivated state. Here, extensive, fully atomistic molecular dynamics and free-energy simulations of the low-K⁺ KcsA structure in an explicit lipid bilayer are performed to evaluate the stability of this structure and the selectivity of its binding sites. We find that the low-K⁺ KcsA structure is stable on the timescale of the molecular dynamics simulations performed, and that ions preferably remain in S1 and S4. In the absence of ions, the selectivity filter evolves toward an asymmetric architecture, as already observed in other computations of the high-K⁺ structure of KcsA and KirBac. The low-K⁺ KcsA structure is not permeable by Na⁺, K⁺, or Rb⁺, and the selectivity of its binding sites is different from that of the high-K⁺ structure.     

Modelling of ion permeation in K+ channels by nonequilibrium molecular dynamics simulations: I. Permeation energetics and structure stability.

Because of the great importance of physiological and pathophysiological processes in which ion channels are involved and because their operation is described by physicochemical laws, there have been many attempts to develop physical models able to describe the membrane permeability and also the structural and functional properties of the channel protein structures. In this study (in two parts) we present a series of simulations on a K+ channel model (KcsA) using Nonequilibrium Molecular Dynamics simulations (NEMD), in order to follow structure stability, permeation energetics and the possibility of obtaining quantitative information about the permeation process using the Linear Response Theory (LRT). On K+ ions were applied external forces to determine them to pass through the channel in a relatively small amount of time, accessible computationally. We ascertained a high resistance of the protein to deformation even in conditions when great forces were applied on ions (the system was far from equilibrium). The estimation of energy profiles in the course of ions passage through the channel demonstrates that these proteins create a conductivity pathway with no energetic barriers for ions movement across the channel (which could be present due to ions dehydration). The dynamic model used demonstrates (as proposed before in the literature after the examination of the static KcsA structure obtained by X-Ray crystallography) that this is due to the interaction of ions with the negatively charged carbonyl oxygens of the main polypeptide chain in the selectivity filter region.     

Molecular dynamics of the KcsA K(+) channel in a bilayer membrane

Molecular dynamics (MD) simulations of an atomic model of the KcsA K(+) channel embedded in an explicit dipalmitoylphosphatidylcholine (DPPC) phospholipid bilayer solvated by a 150 mM KCl aqueous salt solution are performed and analyzed. The model includes the KcsA K(+) channel, based on the recent crystallographic structure of, Science. 280:69-77), 112 DPPC, K(+) and Cl(-) ions, as well as over 6500 water molecules for a total of more than 40,000 atoms. Three K(+) ions are explicitly included in the pore. Two are positioned in the selectivity filter on the extracellular side and one in the large water-filled cavity. Different starting configurations of the ions and water molecules in the selectivity filter are considered, and MD trajectories are generated for more than 4 ns. The conformation of KcsA is very stable in all of the trajectories, with a global backbone root mean square (RMS) deviation of less than 1.9 A with respect to the crystallographic structure. The RMS atomic fluctuations of the residues surrounding the selectivity filter on the extracellular side of the channel are significantly lower than those on the intracellular side. The motion of the residues with aromatic side chains surrounding the selectivity filter (Trp(67), Trp(68), Tyr(78), and Tyr(82)) is anisotropic with the smallest RMS fluctuations in the direction parallel to the membrane plane. A concerted dynamic transition of the three K(+) ions in the pore is observed, during which the K(+) ion located initially in the cavity moves into the narrow part of the selectivity filter, while the other two K(+) ions move toward the extracellular side. A single water molecule is stabilized between each pair of ions during the transition, suggesting that each K(+) cation translocating through the narrow pore is accompanied by exactly one water molecule, in accord with streaming potential measurements (, Biophys. J. 55:367-371). The displacement of the ions is coupled with the structural fluctuations of Val(76) and Gly(77), in the selectivity filter, as well as the side chains of Glu(71), Asp(80), and Arg(89), near the extracellular side. Thus the mechanical response of the channel structure at distances as large as 10-20 A from the ions in the selectivity filter appears to play an important role in the concerted transition.     

Normal-mode refinement of anisotropic thermal parameters for potassium channel KcsA at 3.2 A crystallographic resolution

We report a normal-mode method for anisotropic refinement of membrane-protein structures, based on a hypothesis that the global near-native-state disordering of membrane proteins in crystals follows low-frequency normal modes. Thus, a small set of modes is sufficient to represent the anisotropic thermal motions in X-ray crystallographic refinement. By applying the method to potassium channel KcsA at 3.2 A, we obtained a structural model with an improved fit with the diffraction data. Moreover, the improved electron density maps allowed for large structural adjustments for 12 residues in each subunit, including the rebuilding of 3 missing side chains. Overall, the anisotropic KcsA structure at 3.2 A was systematically closer to a 2.0 A KcsA structure, especially in the selectivity filter. Furthermore, the anisotropic thermal ellipsoids from the refinement revealed functionally relevant structural flexibility. We expect this method to be a valuable tool for structural refinement of many membrane proteins with moderate-resolution diffraction data.     

Absence of ion-binding affinity in the putatively inactivated low-[K+] structure of the KcsA potassium channel

Potassium channels are membrane proteins that selectively conduct K(+) across cellular membranes. The narrowest part of their pore, the selectivity filter, is responsible for distinguishing K(+) from Na(+), and can also act as a gate through a mechanism known as C-type inactivation. It has been proposed that a conformation of the KcsA channel obtained by crystallization in presence of low concentration of K(+) (PDB 1K4D) could correspond to the C-type inactivated state. Here, we show using molecular mechanics simulations that such conformation has little ion-binding affinity and that ions do not contribute to its stability. The simulations suggest that, in this conformation, the selectivity filter is mostly occupied by water molecules. Whether such ion-free state of the KcsA channel is physiologically accessible and representative of the inactivated state of eukaryotic channels remains unclear.     

A set of homology models of pore loop domain of six eukaryotic voltage-gated potassium channels Kv1.1-Kv1.6

Homology models of the pore loop domain of six eukaryotic potassium channels, Kv1.1-Kv1.6, were generated based on the crystallographic structure of KcsA. The results of amino acid sequence alignment indicate that these Kv channels are composed of two structurally and functionally independent domains: the N-terminal 'voltage sensor' domain and the C-terminal 'pore loop' domain. The homology models reveal that the pore loop domains of these Kv channels exhibit similar folds to those of KcsA. The structural features and specific packing of aromatic residues around the selectivity filter of these Kv channels are nearly identical to those of KcsA, whereas most of the structural variations occur in the turret as well as in the inner and outer helices. The distribution of polar and nonpolar side chains on the surfaces of the KcsA and Kv channels reveals that they exhibit a segregation of side chains common to most integral membrane proteins. As the hydrogen bond between Glu71 and Asp80 in KcsA plays an important role in stabilizing the channel, the substituted Val residue in the Kv family corresponding to Glu71 of KcsA stabilizes the channel by making hydrophobic contact with Tyr residue from the signature sequence of the selectivity filter. The homology models of these Kv channels provide particularly attractive subjects for further structure-based studies.     

A computational study of ion binding and protonation states in the KcsA potassium channel

We report results from microscopic molecular dynamics and free energy perturbation simulations of the KcsA potassium channel based on its experimental atomic structure. Conformational properties of selected amino acid residues as well as equilibrium positions of K(+) ions inside the selectivity filter and the internal water cavity are examined. Positions three and four (counting from the extracellular site) in the experimental structure correspond to distinctly separate binding sites for K(+) ions inside the selectivity filter. The protonation states of Glu71 and Asp80, which are close to each other and to the selectivity filter, as well as K(+) binding energies are determined using free energy perturbation calculations. The Glu71 residue which is buried inside a protein cavity is found to be most stable in the neutral form while the solvent exposed Asp80 is ionized. The channel altogether exothermically binds up to three ions, where two of them are located inside the selectivity filter and one in the internal water cavity. Ion permeation mechanisms are discussed in relation to these results.     

K(+) versus Na(+) ions in a K channel selectivity filter: a simulation study

Molecular dynamics simulations of a bacterial potassium channel (KcsA) embedded in a phospholipid bilayer reveal significant differences in interactions of the selectivity filter with K(+) compared with Na(+) ions. K(+) ions and water molecules within the filter undergo concerted single-file motion in which they translocate between adjacent sites within the filter on a nanosecond timescale. In contrast, Na(+) ions remain bound to sites within the filter and do not exhibit translocation on a nanosecond timescale. Furthermore, entry of a K(+) ion into the filter from the extracellular mouth is observed, whereas this does not occur for a Na(+) ion. Whereas K(+) ions prefer to sit within a cage of eight oxygen atoms of the filter, Na(+) ions prefer to interact with a ring of four oxygen atoms plus two water molecules. These differences in interactions in the selectivity filter may contribute to the selectivity of KcsA for K(+) ions (in addition to the differences in dehydration energy between K(+) and Na(+)) and the block of KcsA by internal Na(+) ions. In our simulations the selectivity filter exhibits significant flexibility in response to changes in ion/protein interactions, with a somewhat greater distortion induced by Na(+) than by K(+) ions.     

A comparison between two prokaryotic potassium channels (KirBac1.1 and KcsA) in a molecular dynamics (MD) simulation study

The two potassium ion channels KirBac1.1 and KcsA are compared in a Molecular Dynamics (MD) simulation study. The location and motion of the potassium ions observed in the simulations are compared to those in the X-ray structures and previous simulations. In our simulations several of the crystallography resolved ion sites in KirBac1.1 are occupied by ions. In addition to this, two in KirBac1.1 unresolved sites where occupied by ions at sites that are in close correspondence to sites found in KcsA. There is every reason to believe that the conserved alignment of the selectivity filter in the potassium ion channel family corresponds to a very similar mechanism for ion transport across the filter. The gate residues, Phe146 in KirBac1.1 and Ala111 in KcsA acted in the simulations as effective barriers which never were passed by ions nor water molecules.     

Simulations of ion permeation through a potassium channel: molecular dynamics of KcsA in a phospholipid bilayer

Potassium channels enable K(+) ions to move passively across biological membranes. Multiple nanosecond-duration molecular dynamics simulations (total simulation time 5 ns) of a bacterial potassium channel (KcsA) embedded in a phospholipid bilayer reveal motions of ions, water, and protein. Comparison of simulations with and without K(+) ions indicate that the absence of ions destabilizes the structure of the selectivity filter. Within the selectivity filter, K(+) ions interact with the backbone (carbonyl) oxygens, and with the side-chain oxygen of T75. Concerted single-file motions of water molecules and K(+) ions within the selectivity filter of the channel occur on a 100-ps time scale. In a simulation with three K(+) ions (initially two in the filter and one in the cavity), the ion within the central cavity leaves the channel via its intracellular mouth after approximately 900 ps; within the cavity this ion interacts with the Ogamma atoms of two T107 side chains, revealing a favorable site within the otherwise hydrophobically lined cavity. Exit of this ion from the channel is enabled by a transient increase in the diameter of the intracellular mouth. Such "breathing" motions may form the molecular basis of channel gating.     

The ionization state and the conformation of Glu-71 in the KcsA K(+) channel.

The side chain of Glu-71 of the KcsA K(+) channel, an important residue in the vicinity of the selectivity filter, was not resolved in the crystallographic structure of Doyle et al. (Doyle, D. A., J. M. Cabral, R. A. Pfuetzner, A. Kuo, J. M. Gulbis, S. L. Cohen, B. T. Chait, and R. MacKinnon. 1998. Science. 280:69-77). Its atomic coordinates are undetermined and its ionization state is unknown. For meaningful theoretical and computational studies of the KcsA K(+) channel, it is essential to address questions about the conformation and the ionization state of this residue in detail. In previous MD simulations in which the side chain of Glu-71 is protonated and forming a strong hydrogen bond with Asp-80 it was observed that the channel did not deviate significantly from the crystallographic structure (Bernèche, S., and B. Roux. 2000. Biophys. J. 78:2900-2917). In contrast, we show here that the structure of the selectivity filter of the KcsA channel is significantly disrupted when these side chains are fully ionized on each of the four monomers. To further resolve questions about the ionization state of Glu-71 we calculated the pK(a) value of this residue using molecular dynamics free energy simulations (MD/FES) with a fully flexible system including explicit solvent and membrane and finite-difference Poisson-Boltzmann (PB) continuum electrostatics. It is found that the pK(a) of Glu-71 is shifted by approximately +10 pK(a) units. These results strongly suggest that Glu-71 is protonated under normal conditions.     

Camps 2.0: exploring the sequence and structure space of prokaryotic, eukaryotic, and viral membrane proteins

Structural bioinformatics of membrane proteins is still in its infancy, and the picture of their fold space is only beginning to emerge. Because only a handful of three-dimensional structures are available, sequence comparison and structure prediction remain the main tools for investigating sequence-structure relationships in membrane protein families. Here we present a comprehensive analysis of the structural families corresponding to α-helical membrane proteins with at least three transmembrane helices. The new version of our CAMPS database (CAMPS 2.0) covers nearly 1300 eukaryotic, prokaryotic, and viral genomes. Using an advanced classification procedure, which is based on high-order hidden Markov models and considers both sequence similarity as well as the number of transmembrane helices and loop lengths, we identified 1353 structurally homogeneous clusters roughly corresponding to membrane protein folds. Only 53 clusters are associated with experimentally determined three-dimensional structures, and for these clusters CAMPS is in reasonable agreement with structure-based classification approaches such as SCOP and CATH. We therefore estimate that ～1300 structures would need to be determined to provide a sufficient structural coverage of polytopic membrane proteins. CAMPS 2.0 is available at http://webclu.bio.wzw.tum.de/CAMPS2.0/.     

Pore Hydration States of KcsA Potassium Channels in Membranes

Water-filled hydrophobic cavities in channel proteins serve as gateways for transfer of ions across membranes, but their properties are largely unknown. We determined water distributions along the conduction pores in two tetrameric channels embedded in lipid bilayers using neutron diffraction: potassium channel KcsA and the transmembrane domain of M2 protein of influenza A virus. For the KcsA channel in the closed state, the distribution of water is peaked in the middle of the membrane, showing water in the central cavity adjacent to the selectivity filter. This water is displaced by the channel blocker tetrabutyl-ammonium. The amount of water associated with the channel was quantified, using neutron diffraction and solid state NMR. In contrast, the M2 proton channel shows a V-shaped water profile across the membrane, with a narrow constriction at the center, like the hourglass shape of its internal surface. These two types of water distribution are therefore very different in their connectivity to the bulk water. The water and protein profiles determined here provide important evidence concerning conformation and hydration of channels in membranes and the potential role of pore hydration in channel gating.     

Molecular docking of the scorpion toxin Tc1 to the structural model of the voltage-gated potassium channel Kv1.1 from human Homo sapiens

In this study, structural model of the pore loop region of the voltage-gated potassium channel Kv1.1 from human Homo sapiens was constructed based on the crystallographic structure of KcsA by structural homology. The pore loop region of Kv1.1 exhibits similar folds as that of KcsA. The structural feature of the selectivity filter of Kv1.1 is nearly identical to that of KcsA, whereas most of the structural variations occur in the turret as well as in the inner and outer helices. Molecular docking experiments of the scorpion toxin Tc1 from Tityus cambridgei to the outer vestibule of KcsA as well as Kv1.1 were subsequently performed with various initial Tc1 orientations. Tc1 was found to form the most stable complexes with these two K+ channels when the side chain of Lys14 occupies the pore of the selectivity filter through electrostatic interaction. Tc1 binds preferentially towards Kv1.1 than KcsA due to stronger hydrophobic and electrostatic interactions formed between the toxin and the selectivity filter and outer vestibule of Kv1.1. Furthermore, surface complementarity of the outer vestibules of the channels to the Tc1 spatial conformations also plays an important role in stabilizing both the Tc1/KcsA and Tc1/Kv1.1 complexes.     

Molecular dynamics study of the KcsA potassium channel

The structural, dynamical, and thermodynamic properties of a model potassium channel are studied using molecular dynamics simulations. We use the recently unveiled protein structure for the KcsA potassium channel from Streptomyces lividans. Total and free energy profiles of potassium and sodium ions reveal a considerable preference for the larger potassium ions. The selectivity of the channel arises from its ability to completely solvate the potassium ions, but not the smaller sodium ions. Self-diffusion of water within the narrow selectivity filter is found to be reduced by an order of magnitude from bulk levels, whereas the wider hydrophobic section of the pore maintains near-bulk self-diffusion. Simulations examining multiple ion configurations suggest a two-ion channel. Ion diffusion is found to be reduced to approximately (1)/(3) of bulk diffusion within the selectivity filter. The reduced ion mobility does not hinder the passage of ions, as permeation appears to be driven by Coulomb repulsion within this multiple ion channel.     

NMR study of the tetrameric KcsA potassium channel in detergent micelles

Nuclear magnetic resonance (NMR) studies of large membrane-associated proteins are limited by the difficulties in preparation of stable protein-detergent mixed micelles and by line broadening, which is typical of these macroassemblies. We have used the 68-kDa homotetrameric KcsA, a thermostable N-terminal deletion mutant of a bacterial potassium channel from Streptomyces lividans, as a model system for applying NMR methods to membrane proteins. Optimization of measurement conditions enabled us to perform the backbone assignment of KcsA in SDS micelles and establish its secondary structure, which was found to closely agree with the KcsA crystal structure. The C-terminal cytoplasmic domain, absent in the original structure, contains a 14-residue helix that could participate in tetramerization by forming an intersubunit four-helix bundle. A quantitative estimate of cross- relaxation between detergent and KcsA backbone amide protons, together with relaxation and light scattering data, suggests SDS-KcsA mixed micelles form an oblate spheroid with approximately 180 SDS molecules per channel. K(+) ions bind to the micelle-solubilized channel with a K(D) of 3 +/- 0.5 mM, resulting in chemical shift changes in the selectivity filter. Related pH-induced changes in chemical shift along the "outer" transmembrane helix and the cytoplasmic membrane interface hint at a possible structural explanation for the observed pH-gating of the potassium channel.     

Permeation of water through the KcsA K+ channel

Previous studies have reported that the KcsA potassium channel has an osmotic permeability coefficient of 4.8 x 10(-12) cm3/s, giving it a significantly higher osmotic permeability coefficient than that of some membrane channels specialized in water transport. This high osmotic permeability is proposed to occur when the channel is depleted of potassium ions, the presence of which slow down the water permeation process. The atomic structure of the potassium-depleted KcsA channel and the mechanisms of water permeation have not been well characterized so far. Here, all-atom molecular dynamics simulations, in conjunction with an umbrella sampling strategy and a nonequilibrium approach to simulate pressure gradients are employed to illustrate the permeation of water in the absence of ions through the KcsA K+ channel. Equilibrium molecular dynamics simulations (95 ns combined total length) identified a possible structure of the potassium-depleted KcsA channel, and umbrella sampling calculations (160 ns combined total length) revealed that this structure is not permeable by water molecules moving along the channel axis. The simulation of a pressure gradient across the channel (30 ns combined total length) identified an alternative permeation pathway with a computed osmotic permeability of approximately (2.7 +/- 0.9) x 10(-13) cm3/s. Water fluxes along this pathway did not proceed through collective water motions or transitions to vapor state. All of the major results of this study were robust against variations in a wide set of simulation parameters (force field, water model, membrane model, and channel conformation).