Clare L. Murray

Structure of N-myristoyltransferase with bound myristoylCoA and peptide substrate analogs.

N-myristoyltransferase (Nmt) attaches myristate to the N-terminal glycine of many important eukaryotic and viral proteins. It is a target for anti-fungal and anti-viral therapy. We have determined the structure, to 2.9 Å resolution, of a ternary complex of Saccharomyces cerevisiae Nmt1p with bound myristoylCoA and peptide substrate analogs. The model reveals structural features that define the enzymes substrate specificities and regulate the ordered binding and release of substrates and products. A novel catalytic mechanism is proposed involving deprotonation of the N-terminal ammonium of a peptide substrate by the enzymes C-terminal backbone carboxylate.

The central ‘relay’ unit in hydraphile channels as a model for the water-and-ion ‘capsule’ of channel proteins

The central relay of hydraphile channels is a model for the central ion-capsule of the KcsA K1 K+-conducting channel of Streptomyces lividans; organization of water and concomitant electrostatic stabilization of a transient K+ appear to be the functions in both cases.

A Comparison of Phenyl- and Naphthyl-substituted tris(Macrocycle), Cation-conducting Channels to Assess the Effect of Extended Aromaticity

Abstract Two sodium-cation-conducting, tris(macrocycle) channels, differing only in the size of the terminal aryl groups, have been prepared, characterized, and shown by the planar bilayer conductance method to exhibit qualitative differences in channel activity attributable to the π-electron surface area difference.

Spacer chain length dependence in hydraphile channels: Implications for channel position within phospholipid bilayers

Abstract Five benzyl-terminal tris(macrocycle)s were prepared in an effort to determine the optimal length for hydraphile channels. They are represented in shorthand as PhCH 2 C n C n CH 2 Ph, in which is diaza-18-crown-6 and n is 8 ( 3 ), 10 ( 4 ), 12 ( 5 ), 14 ( 6 ), or 16 ( 7 ). As the length increased by ∼5 A for 6 and 7 (10 A) compared to 5 , it was expected that flux would eventually diminish owing to excessive conformational flexibility. Shortening of the spacer chains by a corresponding increment ( 3 , 4 ) was expected to lead eventually to a compound that was too short and thus incapable of transporting Na + on the timescale of the analytical method. Cation transport rates were measured by dynamic 23 Na NMR and normalized to the activity of Dn C 12 C 12 Dn ( 2 , Dn=dansyl), used as a standard. The observed activity was in the order 5 (C 12 ) ∼ 6 (C 14 )> 4 (C 10 ) ∼ 7 (C 16 ). No cation transport activity was observed for 3 (C 8 ). We estimate the optimal channel length to be ∼45 A, the same as reported for the pore of the KcsA K1 channel.

Crystallographic phasing of myristoyl-CoA-protein N-myristoyltransferase using an iodinated analog of myristoyl-CoA

Myristoyl-CoA-protein N-myristoyltransferase (Nmt; E.C. 2.1.3.97) catalyzes the covalent attachment of myristate to the N-terminal glycine amine of many eukaryotic and viral proteins. The molecular structure of the ternary complex of Saccharomyces cerevisiae Nmt1p with a bound non-hydrolyzable myristoyl-CoA analog, S-(2-oxopentadecyl)-CoA, and a competitive peptidomimetic inhibitor, SC-58272, was solved to 2.9 A resolution by X-ray crystallography. The structure determination utilized diffraction data from an iodinated ternary complex in which a newly designed and synthesized compound, S-(13-iodo-2-oxotridecyl)-CoA, was substituted for S-(2-oxopentadecyl)-CoA. Replacing the two terminal fatty acid C atoms of myristate by iodine produced, under the same crystallization conditions, heavy-atom-derivatized crystals of defined site occupancy that were isomorphous to the native complex. This approach for obtaining experimental phase information can be extended to other crystal structures of protein-fatty acyl complexes. The synthesis of S-(13-iodo-2-oxotridecyl)-CoA and the phasing procedure are described.

Evidence for multiple alkali metal cation complexation in membrane-spanning ion transporters

The polynitrogen-containing cation transporters 1–4 are shown by electrospray mass spectrometry to form multi-cationic species. In the absence of Na+, protonated species dominate but increasing the sodium concentration leads to all binding sites being occupied.

Synthetic, Sodium-Ion-Conducting Tris(Macrocycle) Channels that Function in a Phospholipid Bilayer Membrane: An Overview

Abstract A family of tris(macrocyclic) compounds of the form sidearmCnCnsidearm, has been prepared, characterized chemically, and assayed by dynamic 23Na-NMR for the ability to transport sodium cations in a mixed phosphatidylglycerol/phosphatidylcholine vesicle system. A number of control experiments have been conducted that reinforce the hypothesis that cation transport occurs by a channel mechanism. The tris(macrocycle) channels proved to be functional at a level of 25–50% of that found for the natural pentadecapeptide channel gramicidin. Fluorescence data confirm the expected position of the channel within the bilayer.