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MIT 7 61 - Chemistry of ion coordination and hydration

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NATURE|VOL 414|1 NOVEMBER 2001|www.nature.com 43articlesChemistry of ion coordination andhydration revealed by a K+channel±Fabcomplex at 2.0 AÊresolutionYufeng Zhou, JoaÄo H. Morais-Cabral*, Amelia Kaufman & Roderick MacKinnonHoward 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. Todiscover how the K+channel solves this fundamental aspect of ion conduction, we solved the structure of the KcsA K+channel incomplex with a monoclonal Fab antibody fragment at 2.0 AÊresolution. Here we show how the K+channel displaces watermolecules around an ion at its extracellular entryway, and how it holds a K+ion in a square antiprism of water molecules in a cavitynear its intracellular entryway. Carbonyl oxygen atoms within the selectivity ®lter form a very similar square antiprism aroundeach 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+ionconcentration near the selectivity ®lter varies in response to channel gating.Potassium channels control the electric potential across cellmembranes by catalysing the rapid, selective diffusion of K+ionsdown their electrochemical gradient1. The structure of the K+channel has provided a ®rm basis for understanding the mechan-isms of rapid K+ion transport underlying electrical signalling incells2. Through the interactions of dehydrated K+ions within thechannel's selectivity ®lter, high conduction rates are achieved inthe setting of exquisite ion selectivity3. Two fundamental questionssurrounding this process are addressed in the present study. The®rst is how the K+channel mediates the transfer of a K+ion fromits hydrated state in solution to its dehydrated state in theselectivity ®lter. The issue of dehydration is relevant to allmechanisms of selective ion transport, and because dehydrationin the wrong environment is energetically costly, we should expectto discover in the K+channel a very precise set of mechanismsdesigned to handle hydrated K+ions, and to mediate theirdehydration.The second question addressed in this study is related to thecellular environment in which K+channels operate: inside the cellthe K+concentration is greater than 100 mM, whereas on theoutside the K+concentration is usually less than 5 mM. The K+channel gate, or door that opens and closes the pore, is locatedbetween 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 isexposed to a low K+concentration from outside. This is aninteresting situation when one considers the structure of theselectivity ®lter, and the mechanism by which it conducts K+ions.The ®lter points a large number of carbonyl oxygen atoms into thepore. Owing to the partial negative charge on these atoms, thiswould be an unlikely structure if it were not for the presence ofdehydrated K+ions in the ®lter. In other words, the K+ions that gothrough the ®lter are actually counter-charges, necessary for itsstructure. The question that then arises is what happens when thechannel's gate closes and the selectivity ®lter is in equilibrium with alow extracellular K+environment. We answer this by describing thestructure of the K+selectivity ®lter in the presence of a low K+ionconcentration.K+channel±Fab complexTo address the above questions it was necessary to solve the K+channel structure at a resolution that would reveal ordered watermolecules and protein chemistry with high accuracy. To achieve thisend, we raised monoclonal antibodies against the KcsA K+channeland selected clones on the basis of their ability to recognize thetetrameric but not the monomeric form of the channel7,8.AK+channel±Fab complex with a stoichiometry of one Fab fragment perchannel 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 AÊBragg spacings at thesynchrotron. We solved phases by molecular replacement using apublished Fab structure (Protein Data Bank (PDB) code 1MLC)9,and could easily interpret the resulting electron density map. Thepublished KcsA K+channel structure (PDB code 1BL8) was placedinto the density map2, followed by several cycles of rebuilding andre®nement. The ®nal model, referred to as the high-K+structure(200 mM K+), is re®ned with good stereochemistry to an Rfand Rwof 23.3% and 21.8%, respectively, and contains 534 amino acids, 7K+ions, 469 water molecules and 2 partial lipids. A secondstructure, the low-K+structure (3 mM K+), was solved at 2.3 AÊresolution to an Rfand Rwof 23.5% and 21.8%, respectively, andcontains 534 amino acids, 2 K+ions, 1 Na+ion, 266 water moleculesand 2 partial lipids (Table 1).The Fab fragment is attached to the K+channel turret on theextracellular face of the channel (Fig. 1). All of the protein contactswithin the crystal are formed between neighbouring Fab fragments,with the K+channel conveniently suspended so that its detergentmicelle is not involved in crystal contacts. This packing arrangementundoubtedly accounts for the high-quality X-ray diffraction7,8.Furthermore, Fab attachment to the turrets leaves open a widepassageway outside the pore, so that ion binding should beunperturbed 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 atfour K+ion-binding sites inside the selectivity ®lter, correspondingto positions 1±4 from outside the cell to inside. Strong electron* Present address: Department of Molecular Biophysics and Biochemistry, Yale University, 260 WhitneyAvenue, New Haven, Connecticut 06520, USA.© 2001 Macmillan Magazines Ltddensity is also visible at both entryways to the ®lter, curiouslysuspended along the pore


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