KcsA

Procarboxypeptidase A is activated by the removal of a peptide of some 64 residues from the N-terminus by trypsin.153 This zymogen has significant catalytic activity. As well as catalyzing the hydrolysis of small esters and peptides, procarboxypeptidase removes the C-terminal leucine from lysozyme only seven times more slowly than does carboxypeptidase. Also, the zymogen hydrolyzes Bz-Gly-L-Phe with kcsA = 3 s-1 and KM = 2.7 mM, compared with values of 120 s 1 and 1.9 mM for the reaction of the enzyme.154 In contrast to the situation in chymotrypsinogen, the binding site clearly pre-exists in procarboxypeptidase, and the catalytic apparatus must be nearly complete. 

The QM/MM and ab initio methodologies have just begun to be applied to challenging problems involving ion channels [73] and proton motion through them [74]. Reference [73] utilizes Hartree-Fock and DFT calculations on the KcsA channel to illustrate that classical force fields can fail to include polarization effects properly due to the interaction of ions with the protein, and protein residues with each other. Reference [74] employs a QM/MM technique developed in conjunction with Car-Parrinello ab initio simulations [75] to model proton and hydroxide ion motion in aquaporins. Due to the large system size, the time scale for these simulations was relatively short (lOps), but the influences of key residues and macrodipoles on the short time motions of the ions could be examined. 

Bliznyuk, A. A. Rendell, A. P., Electronic effects in biomolecular simulations investigations of the KcsA potassium ion channel, J. Phys. Chem. B 2004,108, 13866-13873... 

Roux, B. MacKinnon, R., The cavity and pore helices in the KcsA K+ channel electrostatic stabilization of monovalent cations, Science 1999, 285, 100-102... 

Burykin, A. Kato, M. Warshel, A., Exploring the origin of the ion selectivity of the KcsA potassium channel, Proteins 2003, 52, 412-426. 

We end this section with an example of a recent MD study of Tieleman and co-workers [168] on the selectivity of a bacterial K+ channel, KcsA, which is formed by a protein. 

KcsA, as used by Shrivastava et al. [168], The channel consists of four peptide chains (for clarity in the picture only two are given), and is embedded in a lipid bilayer. The structure can be thought of as being made up of a selectivity filter, a central cavity, and a gate at the inner side of the bilayer, (b) Water molecules and K+ ions in the selectivity filter SO represents the extracellular mouth. From [168]. Reproduced by permission of the Biophysical Society... 

Figure 1.3 Selective-binding sites in transport proteins for Na+, K+, Ca2+ and Cl. (a) Two Na+ binding sites in the LeuT Na+-dependent pump, (b) Four K+ binding sites in the KcsA K+ channel, (c) Two Ca2+ binding sites in the Ca2+ ATPase pump, (d) Two central Cl binding sites in a mutant C1C Cl /H+ exchanger. (From Gouax and MacKinnon, 2005. Copyright (2005) American Association for the Advancement of Science.)...

Figure 9.2 (a) The structure of the KcsA K+ channel with two of the four subunits removed the pore helices are in red, the selectivity filter in yellow the electron density along the ion pathway is in blue mesh, (b) The K+-selectivity filter (two subunits) with eight carbonyl groups (red) coordinating K+ ions (green spheres) in positions 1-4 from the extracellular side. (From MacKinnon, 2004. Reproduced with permission from John Wiley Sons., Inc.)... 

Figure 4.3 Sequence alignment of hERG and other representative l<+ channels alignments of the pore helix, selectivity filter and inner helices. The inner helix of KcsA corresponds with S6 of the other l<+ channels shown here, which are all voltage-gated channels. hERG residues close to the selectivity filter and on S6 implicated in drug...

In Figure 5 of reference 16, the pore region is based on the KcsA K channel from PDB 1BL8. Co-crystallized with Fab monoclonal antibodies from mouse. 

The Shaker potassium channel did not yield crystals for the first potassium ion channel X-ray crystallographic structure. Rather, it was the prokaryote bacterium Streptomyces lividans, abbreviated as the KcsA K+ channel, that first produced crystals suitable for crystallography. This structure, published in 1998 by MacKinnon s group in Science magazine, was received with great praise from the scientific community. It was known at that time that the amino acid sequence of KcsA was similar to that of other K channels, including vertebrate and invertebrate voltage-dependent K (Kv) channels (such as the... 

KcsA crystals suitable for X-ray crystallographic analysis using synchrotron radiation were obtained and the data collected and analyzed for multiple crystals and six different data sets as described in the 1998 Science publication (reference 15). The final KcsA pore structure, including amino acid residues 23 to 119 of the K+ channel, refined to 3.2 A. The X-ray data were deposited in the Protein Data Bank with the accession number 1BL8. 

---