Liu, Y.-S., Sompornpisut, P. and Perozo, E. Structure of the KcsA channel intracellular gate in the open state. Nature Struct. Biol. 8, 883-887 (2001).
Ion channels catalyze the selective transfer of ions across the membrane in response to a variety of stimuli. These channels gate by controlling the access of ions to a centrally located water-filled pore. The crystal structure of the Streptomyces lividans potassium channel (KcsA) has allowed a molecular exploration of this mechanism. Electron paramagnetic resonance (EPR) studies have uncovered significant conformational changes at the intracellular end of the second transmembrane helix (TM2) upon gating. We have used site-directed spin labeling (SDSL) and EPR spectroscopy in an attempt to quantify the structural rearrangements of the KcsA TM2 bundle underlying the transition from the closed to the open state. Under conditions favoring the closed and open conformations, 10 intersubunit distances were obtained across TM2 segments from tandem dimer constructs. Analysis of these data points to a mechanism in which each TM2 helix tilts away from the permeation pathway, towards the membrane plane, and rotates about its helical axis, supporting a scissoring-type motion with a pivot point near residues 107-108. These movements are accompanied by a large increase in the diameter of the vestibule below the central water-filled cavity.
Sompornpisut, P., Liu, Y.-S. and Perozo, E. Calculation of rigid-body conformational changes using restraint-driven Cartesian transformations. Biophys. J. 81, 2530-2546(2001).
We present an approach for calculating conformational changes in membrane proteins using limited distance information. The method, named restraint-driven Cartesian transformations, involves 1) the use of relative distance changes; 2) the systematic sampling of rigid body movements in Cartesian space; 3) a penalty evaluation; and 4) model refinement using energy minimization. As a test case, we have analyzed the structural basis of activation gating in the Streptomyces lividans potassium channel (KcsA). A total of 10 pairs of distance restraints derived from site-directed spin labeling and electron paramagnetic resonance (SDSL-EPR) spectra were used to calculate the open conformation of the second transmembrane domains of KcsA (TM2). The SDSL-EPR based structure reveals a gating mechanism consistent with a scissoring-type motion of the TM2 segments that includes a pivot point near middle of the helix. The present approach considerably reduces the amount of time and effort required to establish the overall nature of conformational changes in membrane proteins. It is expected that this approach can be implemented into restrained molecular dynamics protocol to calculate the structure and conformational changes in a variety of membrane protein systems.
Patel, H.V., Vyas, A.A., Vyas, K.A., Liu, Y.-S., Chiang, C.-M., Chi, L.M. and Wu, Wg. Heparin and heparan sulfate bind to snake cardiotoxin. Sulfated oligosaccharides as a potential target for cardiotoxin action. J. Biol. Chem. 272, 1484-1492 (1997).
Cardiotoxins (CTXs) from
cobra venom show cytotoxicity toward several cell types. They cause systolic
heart arrest and severe tissue necrosis. Their interaction with phospholipids
is established but by itself fails to explain the specificity of these
toxins; other component(s) of membrane must, therefore, intervene to direct
them toward their target. We herein show, for the first time, that sulfated
glycosaminoglycans, heparin, heparan sulfate (HS), chondroitin sulfate
(CS), and dermatan sulfate (DS), interact with CTX A3, a major component
of Taiwan cobra venom, by use of affinity chromatography, circular dichroism,
absorbance, and fluorescence intensity and anisotropy measurements. The
relative strength of binding, determined by the NaCl concentration required
to dissociate the CTX-glycosaminoglycan complex, varied as follows: heparin
> DS > CS > HS. In physiological buffer (8 mM Na2HPO4, 2.7 mM KCl, 1.8
mM KH2PO4, 138 mM NaCl, pH 7.4), however, only heparin and HS bound to
CTX, with respective dissociation constants of 1.4 and 16 microM, while
CS and DS failed to exhibit well defined binding behavior, as indicated
by fluorescence measurements. We estimate that CTX makes 3-4 ionic contacts
with heparin based on a salt-dependent binding study and that approximately
40% of binding free energy is derived from purely electrostatic interactions
under physiological conditions. Sulfated pentasaccharide may be sufficient
to bind to CTX. We also found that heparin accentuates the penetration
of CTX into phospholipid membranes as analyzed by Langmuir monolayer measurement.
In view of these results we propose that heparin-like moieties of the cell
surface may modulate the action of CTX.