MIT 7 61 - Chemistry of ion coordination and hydration (6 pages)

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Chemistry of ion coordination and hydration



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Chemistry of ion coordination and hydration

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6
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Massachusetts Institute of Technology
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7 61 - Eukaryotic Cell Biology: Principles and Practice
Eukaryotic Cell Biology: Principles and Practice Documents
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articles Chemistry of ion coordination and hydration revealed by a K channel Fab complex at 2 0 A resolution Yufeng Zhou Joa o H Morais Cabral Amelia Kaufman Roderick MacKinnon Howard Hughes Medical Institute Laboratory of Molecular Neurobiology and Biophysics Rockefeller University 1230 York Avenue New York New York 10021 USA Ion transport proteins must remove an ion s hydration shell to coordinate the ion selectively on the basis of its size and charge To discover how the K channel solves this fundamental aspect of ion conduction we solved the structure of the KcsA K channel in complex with a monoclonal Fab antibody fragment at 2 0 A resolution Here we show how the K channel displaces water molecules around an ion at its extracellular entryway and how it holds a K ion in a square antiprism of water molecules in a cavity near its intracellular entryway Carbonyl oxygen atoms within the selectivity lter form a very similar square antiprism around each K binding site as if to mimic the waters of hydration The selectivity lter changes its ion coordination structure in low K solutions This structural change is crucial to the operation of the selectivity lter in the cellular context where the K ion concentration near the selectivity lter varies in response to channel gating Potassium channels control the electric potential across cell membranes by catalysing the rapid selective diffusion of K ions down their electrochemical gradient1 The structure of the K channel has provided a rm basis for understanding the mechanisms of rapid K ion transport underlying electrical signalling in cells2 Through the interactions of dehydrated K ions within the channel s selectivity lter high conduction rates are achieved in the setting of exquisite ion selectivity3 Two fundamental questions surrounding this process are addressed in the present study The rst is how the K channel mediates the transfer of a K ion from its hydrated state in solution to its dehydrated state in the selectivity lter The issue of dehydration is relevant to all mechanisms of selective ion transport and because dehydration in the wrong environment is energetically costly we should expect to discover in the K channel a very precise set of mechanisms designed to handle hydrated K ions and to mediate their dehydration The second question addressed in this study is related to the cellular environment in which K channels operate inside the cell the K concentration is greater than 100 mM whereas on the outside the K concentration is usually less than 5 mM The K channel gate or door that opens and closes the pore is located between the selectivity lter and the intracellular solution2 4 6 Therefore when the gate is open the lter is exposed to a high K concentration from inside the cell and when it is closed the lter is exposed to a low K concentration from outside This is an interesting situation when one considers the structure of the selectivity lter and the mechanism by which it conducts K ions The lter points a large number of carbonyl oxygen atoms into the pore Owing to the partial negative charge on these atoms this would be an unlikely structure if it were not for the presence of dehydrated K ions in the lter In other words the K ions that go through the lter are actually counter charges necessary for its structure The question that then arises is what happens when the channel s gate closes and the selectivity lter is in equilibrium with a low extracellular K environment We answer this by describing the Present address Department of Molecular Biophysics and Biochemistry Yale University 260 Whitney Avenue New Haven Connecticut 06520 USA NATURE VOL 414 1 NOVEMBER 2001 www nature com structure of the K selectivity lter in the presence of a low K ion concentration K channel Fab complex To address the above questions it was necessary to solve the K channel structure at a resolution that would reveal ordered water molecules and protein chemistry with high accuracy To achieve this end we raised monoclonal antibodies against the KcsA K channel and selected clones on the basis of their ability to recognize the tetrameric but not the monomeric form of the channel7 8 A K channel Fab complex with a stoichiometry of one Fab fragment per channel subunit was produced and crystallized in space group I4 with one channel subunit and Fab fragment per asymmetric unit Frozen crystals diffracted X rays to 2 0 A Bragg spacings at the synchrotron We solved phases by molecular replacement using a published Fab structure Protein Data Bank PDB code 1MLC 9 and could easily interpret the resulting electron density map The published KcsA K channel structure PDB code 1BL8 was placed into the density map2 followed by several cycles of rebuilding and re nement The nal model referred to as the high K structure 200 mM K is re ned with good stereochemistry to an Rf and Rw of 23 3 and 21 8 respectively and contains 534 amino acids 7 K ions 469 water molecules and 2 partial lipids A second structure the low K structure 3 mM K was solved at 2 3 A resolution to an Rf and Rw of 23 5 and 21 8 respectively and contains 534 amino acids 2 K ions 1 Na ion 266 water molecules and 2 partial lipids Table 1 The Fab fragment is attached to the K channel turret on the extracellular face of the channel Fig 1 All of the protein contacts within the crystal are formed between neighbouring Fab fragments with the K channel conveniently suspended so that its detergent micelle is not involved in crystal contacts This packing arrangement undoubtedly accounts for the high quality X ray diffraction7 8 Furthermore Fab attachment to the turrets leaves open a wide passageway outside the pore so that ion binding should be unperturbed by the presence of the Fab fragments An electron density map surrounding the channel s selectivity lter in the high K structure is shown in Fig 2 Density is present at four K ion binding sites inside the selectivity lter corresponding to positions 1 4 from outside the cell to inside Strong electron 2001 Macmillan Magazines Ltd 43 articles Table 1 Crystallographic analysis Data set Data collected Resolution A Redundancy overall outer Completeness overall outer Rsymm overall outer 99 1 95 2 99 2 95 3 0 071 0 530 0 056 0 370 I j overall outer Re ections with I j 2 overall outer High K Low K 50 2 0 30 2 3 3 8 3 0 4 2 2 7 22 4 2 3 24 5 2 2 79 45 80 40 Re nement Resolution A Rw Rf 40 2 0 30 2 3 21 8 23 3 21 8 23 5 Root mean square difference Mean B factor A 2 Bond


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